Method for producing casein and uses thereof

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, and its use in particular for producing cheese replicas.

Description

[Technical field]

[0001] The present invention relates to the food industry and to a novel process for producing a casein composition and in particular its use for producing cheese replicas, in particular vegan cheese. [Background technology]

[0002] The use of milk as a nutrient rich in proteins, sugars, and lipids has become almost universal in previous societies. Moreover, the conversion of milk into various derivatives is one of the oldest examples of human agricultural industry. A wide variety of cheeses are made in every corner of the planet and in some countries it has become a cultural standard, used as such or as an ingredient in many dishes; the use of cheese on pizza, burgers, or as an additive to pasta are many examples of international standards. Today, the cheese market has 20 million tons of product per year, worth approximately $140 billion.

[0003] However, dairy products are associated with several issues or concerns, not only in terms of health but also environmental and ethical considerations, which highlight the need for dairy alternatives that alleviate various issues. Health problems 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 high saturated fatty acid content, which are known to have 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, an enzyme that breaks down lactose in the stomach and small intestine, resulting in 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). It is a very common characteristic in humans, genetically determined, and requires a dairy-free diet. In dairy substitutes, the composition can be adjusted to avoid lactose and saturated fatty acids, as well as allergenic proteins.

[0004] Furthermore, environmental and ethical concerns related to foods of animal origin have been growing over recent decades. The burden of animal reproduction on a population of 7 billion (11 billion in 2050) is becoming ever greater. The environmental impacts are serious. These are currently considered mainly in terms of anthropogenic greenhouse gas (GHG) emissions, water consumption, wastewater pollution and land occupation.

[0005] Livestock farming is considered today one of the leading sources of GHG emissions, estimated at 7.1 gigatons of CO2 equivalent per year, which represents 14.5% of all anthropogenic greenhouse gas (GHG) emission sources and processes (Rotz (2017) Modeling greenhouse gas emissions from dairy farms. J Dairy Science, Volume 101, pages 6675-6690), while a 2010 United Nations study estimated that the dairy sector alone contributes 4% of anthropogenic GHG emissions worldwide (FOOD AND AGRICULTURE ORGANIZATION OF THE UNITED NATIONs, Greenhouse Gas Emissions from the Dairy Sector. A Life Cycle Assessment).

[0006] Very large amounts of water consumption are also associated with livestock production (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 runoff also has a significant impact on the environment. While the impact of crop fertilizers on groundwater should not be ignored, the impact of livestock runoff is often significant and has proven to have disastrous consequences in many parts of the world (https: / / www.nrdc.org / issues / livestock-production). Furthermore, livestock occupy up to nearly 80% of the world's agricultural land, but produce less than 20% of the world's calorie supply. This indicates that livestock production imposes very high levels of stress on land resources.

[0007] Finally, animal welfare is an increasingly important concern as meat and dairy production and processing have become intensive industrial processes that are increasingly recognised as ethically unacceptable due to their large scale.

[0008] Therefore, there is a strong need for dairy alternatives that alleviate the above problems. As a large proportion of milk is converted into cheese (around 38% in Europe), this need accounts for a large part of the need for cheese alternatives.

[0009] Plant-based alternatives offer the potential to replace traditional dairy products. However, these products are often derived from soy, almond or coconut milk and can be far from mimicking the taste of dairy products. Moreover, they are in any case radically different in terms of composition.

[0010] Therefore, today, (i) is free of undesirable compounds; (ii) is similar to the original product in appearance, texture and taste; (iii) Nutritionally equivalent to or greater than the original product There is a need for dairy alternatives, particularly cheese alternatives.

[0011] A typical composition of cow's milk is shown in Table 1. More detailed composition of cow's milk and other animal milks, including listing of various lipids, proteins, salts, vitamins and other nutrients, can be found in a number of 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 (except lactose) can be recovered from plants, calcium from inorganic sources, animal sources or plants (such as seaweed). In the case of protein, other sources must be considered, since animal proteins differ from plant proteins in their amino acid content. Milk proteins are also produced by fermentation, another source of ingredients of non-animal origin.

[0014] Additionally, proteins isolated from milk are also used individually as dietary supplements and other applications. Thus, milk proteins produced by fermentation, separate from other milk components, can be used in applications other than producing dairy alternatives.

[0015] Fermentative production is based on the growth of bacteria or fungi that produce the compound of interest in a fermenter, usually followed by recovery and purification of the compound of interest. The production of proteins by fermentation is a process used in the food industry, one of the first examples being recombinant chymosin, the first artificially produced enzyme registered and approved by the US Food and Drug Administration, which today represents a large part of the rennet market (today more than 80% in the United States) (Food Biotechnology in the United States: Science, Regulation, and Issues”. USDepartment of State.Retrieved 2006-08-14). Since then, several studies have described the production of milk constituent proteins or their homologs by fermentation in various microorganisms (see below), and the assembly of components containing fermented proteins to create milk substitutes has also been described several times (US Pat. No. 6,270,827; US Pat. No. 5,942,274; WO 2018 / 039632; WO 2020 / 223700).

[0016] However, industrial production of milk proteins also requires adapted purification procedures that not only produce the required proteins at a high level of purity at a grade suitable for use in the food industry, but are also easily scalable at industrial levels and at a reasonable cost to develop. Ideally, such procedures should be as simple as possible and avoid the use of non-food chemicals and steps that are expensive to carry out on a very large scale.

[0017] The four casein molecules (alpha-S1-, alpha-S2-, beta- or kappa-casein) represent more than 80% of milk proteins and almost all cheese proteins, since other milk proteins such as beta-lactoglobulin, alpha-lactalbumin, bovine serum albumin and immunoglobulins (commonly called "whey proteins") are removed in the whey after coagulation. Casein solubility is highly dependent on pH, temperature and salt concentration (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), but in milk it is not found as a soluble protein, but is organized in micelles, which result in the formation of colloidal structures. Casein micelles are nearly spherical particles with diameters ranging from 50 to 600 nm, with an average diameter of about 200 nm (Kruif, Supra-aggregates of casein micelles as a prelude to coagulation (1998) J Dairy Sci, Volume 81 pages 3019-3028;de Kruif et al. Casein micelles and their internal structure (2012) Advances in Colloid and Interface Science, Volume 171-172,pages 36-52). The evolution of these colloids into curds is the basis for cheesemaking (Gillis JC, Ayerbe A, Le fromage 4th edition, Lavoisier-Technique Et Documentation 20 avril 2018). κ-casein is thought to play an important role as a stabilizer of the micellar structure.

[0018] Casein can be isolated from milk as sodium, calcium or potassium caseinate by acid-mediated or rennet-mediated coagulation followed by neutralization (Sarode et al. (2016) Methods of Manufacture. In: Caballero, B. Finglas, P. and Toldra, F. (eds.) The Encyclopedia of Food and Health vol. 1, pp. 676-682. Oxford: Academic Press). The different individual caseins (alpha-S1-, alpha-S2-, beta- or kappa-casein) were purified by membrane filtration at low temperatures (Murphy and Fox (1991) Fractionation of sodium caseinate by ultrafiltration. Food Chem. Volume 39 Pages 27-38:;Ward and Bastian(1996)A method for isolating β-casein. J. Dairy Sci. Volume 79,pages 1332-1339;Huppertz et al. (2006). A method for the large-scale isolation of β-casein. Food Chem. Volume 99, pages 45-50;Lamotheet al.(2007).Short communication: Extraction of β-casein from goat milk. J. Dairy Sci. Volume 90, pages 5380-5382;O'Mahony, et al.(2007).Purification of β-casein from milk.; U.S. Patent Publication No. 0104847) or selective precipitation (Law and Leaver (2007) Methods of extracting casein fractions from milk and caseinates and production of novel products. Hanna Research Institute, assignee.; WO 03 / 003847; Post et al.(2009).β-Casein as a bioactive precursor-Processing for purification. Aust.J.Dairy Technol. Volume 64, pages 84-88;Post and Hinrichs(2011)Large-scale isolation of food-grade β-casein. Milchwissenschaft, Volume 66, pages 361-364) can also be used to purify individual casein preparations based on their different physicochemical properties.

[0019] In the case of recombinant proteins produced by fermentation, specific procedures need to be developed to isolate the recombinant protein from the biomass and culture broth, and such procedures depend on the producing microorganism and the properties of the recombinant protein.

[0020] E. coli, one of the mainstays of protein production Escherichia coliIn Escherichia coli, recombinant proteins are often found in inclusion bodies. These intracellular particles consist essentially of aggregates of recombinant proteins. Recovering properly folded proteins from such aggregates requires a laborious refolding process (Singh et al. (2015) Protein recovery from inclusion bodies of Escherichia coli using mild solubilization process. Microbial Cell Factories, Volume 14:pages 41-51;Vallejo and Rinas (2004) Strategies for the recovery of active proteins through refolding of bacterial inclusion body proteins. Microbial Cell Factories Volume 3: pages 11). However, inclusion bodies are small particles (0.2-1.5 mm) and protocols for their isolation are often based on centrifugation at high relative centrifugal forces, a process that is not easy to perform on an industrial scale (Rodriguez-Carmona et al. (2010) Isolation of cell-free bacterial inclusion bodies. Microbial Cell Factories Volume 9, pages 71), which can be problematic for large batches.

[0021] For thermostable recombinant proteins or peptides, the use of thermal lysis can be an interesting purification step, since many of the soluble host proteins precipitate and / or degrade at high temperatures (Takesawa et al. (1990) Heat-induced precipitation of cell homogenates: an investigation of the recovery of thermostable proteins. Enzyme Microb. Technol. Volume 12, pages 184-189; Kirk and Cowan 1995 Optimising the recovery of recombinant thermostable proteins expressed in mesophilic hosts. J. of Biotechnology Volume 42, pages 177-184; Sundarrajan et al. (2018) Novel properties of recombinant Sso7d-Taq DNA polymerase purified using aqueous two-phase extraction: Utilities of the enzyme in viral diagnosis. Biotechnol Rep (Amst) 19:e00270; US Pat. No. 8,603,782; WO 2011 / 119703). Summary of the Invention

[0022] Post et al (Journal of Dairy Science, vol.95, no.4,2012, 1603-1616) is not concerned with the purification of casein but analyzes the reasons for the existence of selective precipitation of beta-casein relative to alpha-casein. The information provided in this document is therefore not relevant to the technical problems of isolating casein, especially recombinant casein.

[0023] US Patent No. 4,550,028 does not describe the use of recombinant caseins or their preparation or purification. It should be noted that this document (column 3, lines 1-22) refers to casein aggregation at acidic pH with elevated temperature. This is different from the technical effect achieved here (casein remains in solution and other proteins that are not milk proteins are precipitated).

[0024] WO 99 / 54355 describes the purification and recovery of recombinant proteins from cells using water-miscible organic solvents (particularly acetone, isopropanol, ethanol and methanol) at a pH of about 5.0 to about 10.0 and at temperatures of about 30° C. to about 50° C. The application states that "the water-miscible organic solvent extracts the recombinant protein from the cell suspension. The water-miscible organic solvent is believed to selectively partition the recombinant protein from the cell suspension into a liquid phase" (page 6, lines 20-24). The objective is similar to that of the present application (purification of recombinant protein), but the technical effect is obtained in a different way (use of organic solvent) than disclosed herein.

[0025] Indeed, in food applications it is important to avoid the use of chemicals such as organic solvents that need to be removed in subsequent steps, and the process disclosed herein is carried out on a bacterial culture medium that is free of organic solvents (preferably after cell lysis and removal of cell debris).

[0026] WO 2020 / 223700 relates to the preparation of recombinant casein-based formulations (alpha and kappa caseins in micellar form, preferably without beta casein as indicated in paragraph

[0006] ). It is noted that this document does not describe the purification of these recombinant proteins, nor their use, and the examples relate to the manufacture of fresh products made from micellar casein, as seen in paragraph

[0210] , "typically obtained in the industry by ultrafiltration of skim milk to isolate the casein micelles and spray drying techniques to powder the casein micelles" or obtained after micellar reconstitution (Example 6).

[0027] US Patent No. 4,519,945 relates to the preparation of curd from milk by adjusting the pH and temperature, thus the document is concerned with the conditions for obtaining the coagulation of casein and not with the objective of isolating and purifying casein.

[0028] The applicant has shown that casein solubilizes or remains soluble at high temperatures, whereas other proteins precipitate and / or degrade. In particular, the applicant has found that in E. coli, recombinant caseins (particularly alpha and beta caseins) found in the insoluble fraction are solubilized by heating at high temperatures (above 75° C.), whereas, in contrast, many soluble proteins from the host are precipitated and / or degraded at such temperatures. Using such properties, it is possible to eliminate all or part of the non-casein proteins in a composition, thus concentrating casein in the composition (i.e. increasing its relative amount). In this way, a process for isolating casein from bacterial cells has been developed, which forms the basis of the invention disclosed herein. [Brief description of the drawings]

[0029] [Figure 1A] Analysis by SDS-PAGE of soluble and insoluble fractions of extracts from recombinant cells expressing alpha-S1 casein (A), alpha-S2 casein (B) and beta casein (C). Cells transformed with vectors expressing casein (BL21-alpha-S1 casein, BL21-alpha-S2 casein or BL21-beta casein) or with empty vector (BL21) were lysed by two different protocols as described in Example 1. Lysates were centrifuged and analyzed by SDS-PAGE. T: total (before centrifugation), S: supernatant, P: pellet. [Figure 1B]Analysis by SDS-PAGE of soluble and insoluble fractions of extracts from recombinant cells expressing alpha-S1 casein (A), alpha-S2 casein (B) and beta casein (C). Cells transformed with vectors expressing casein (BL21-alpha-S1 casein, BL21-alpha-S2 casein or BL21-beta casein) or with empty vector (BL21) were lysed by two different protocols as described in Example 1. Lysates were centrifuged and analyzed by SDS-PAGE. T: total (before centrifugation), S: supernatant, P: pellet. [Figure 1C] Analysis by SDS-PAGE of soluble and insoluble fractions of extracts from recombinant cells expressing alpha-S1 casein (A), alpha-S2 casein (B) and beta casein (C). Cells transformed with vectors expressing casein (BL21-alpha-S1 casein, BL21-alpha-S2 casein or BL21-beta casein) or with empty vector (BL21) were lysed by two different protocols as described in Example 1. Lysates were centrifuged and analyzed by SDS-PAGE. T: total (before centrifugation), S: supernatant, P: pellet. [Figure 2A] SDS-PAGE analysis of soluble and insoluble fractions of heat-treated extracts from recombinant cells expressing alpha-S1 casein (A), alpha-S2 casein (B) and beta casein (C). Cells transformed with vectors expressing casein (BL21-alpha-S1 casein, BL21-alpha-S2 casein or BL21-beta casein) or with an empty vector (BL21) were heated for 10, 20, 30, 60, 90 or 120 min (10'; 20'; 30'; 60'; 90'; 120'; 0': non-heated sample; C: sample incubated at room temperature for 120 min). Lysates were centrifuged and analyzed by SDS-PAGE. T: total (before centrifugation), S: supernatant, P: pellet. For details, see Example 2. [Figure 2B]SDS-PAGE analysis of soluble and insoluble fractions of heat-treated extracts from recombinant cells expressing alpha-S1 casein (A), alpha-S2 casein (B) and beta casein (C). Cells transformed with vectors expressing casein (BL21-alpha-S1 casein, BL21-alpha-S2 casein or BL21-beta casein) or with an empty vector (BL21) were heated for 10, 20, 30, 60, 90 or 120 min (10'; 20'; 30'; 60'; 90'; 120'; 0': non-heated sample; C: sample incubated at room temperature for 120 min). Lysates were centrifuged and analyzed by SDS-PAGE. T: total (before centrifugation), S: supernatant, P: pellet. For details, see Example 2. [Figure 2C] SDS-PAGE analysis of soluble and insoluble fractions of heat-treated extracts from recombinant cells expressing alpha-S1 casein (A), alpha-S2 casein (B) and beta casein (C). Cells transformed with vectors expressing casein (BL21-alpha-S1 casein, BL21-alpha-S2 casein or BL21-beta casein) or with an empty vector (BL21) were heated for 10, 20, 30, 60, 90 or 120 min (10'; 20'; 30'; 60'; 90'; 120'; 0': non-heated sample; C: sample incubated at room temperature for 120 min). Lysates were centrifuged and analyzed by SDS-PAGE. T: total (before centrifugation), S: supernatant, P: pellet. For details, see Example 2. [Figure 3A] SDS-PAGE analysis of beta-casein produced in E. coli in a 42 L fermenter. A. Analysis of total extract. T: Total extract (heated at 95° C. for 5 min in Laemmli loading buffer). C: Beta-casein sigma (5, 10 and 20 μg of protein were loaded in three different lanes). For details, see Example 3. B. Analysis of soluble and insoluble fractions. For details, see Example 3. [Figure 3B]SDS-PAGE analysis of beta-casein produced in E. coli in a 42 L fermenter. A. Analysis of total extract. T: Total extract (heated at 95° C. for 5 min in Laemmli loading buffer). C: Beta-casein sigma (5, 10 and 20 μg of protein were loaded in three different lanes). For details, see Example 3. B. Analysis of soluble and insoluble fractions. For details, see Example 3. [Figure 4A] SDS-PAGE analysis of soluble and insoluble fractions of heat-treated extracts from recombinant cells expressing beta-casein grown in 42 L bioreactors. For details, see Example 4. A. Cells transformed with a vector expressing beta-casein were heated at 95°C for 30, 60, 90 or 120 min (30';60';90';120'). Lysates were centrifuged and soluble and insoluble fractions were analyzed by SDS-PAGE. S: Supernatant; P: Pellet. B. Cells transformed with a vector expressing beta-casein were heated at 55°C for 30, 60, 90 or 120 min (30';60';90';120') or at 95°C for 10, 20, 30, 60, 90 or 120 min (10;20;30';60';90';120'). The lysates were centrifuged and both soluble and insoluble fractions were analyzed by SDS-PAGE. 0': non-heated sample; C: sample incubated at room temperature for 120 min. [Figure 4B]SDS-PAGE analysis of soluble and insoluble fractions of heat-treated extracts from recombinant cells expressing beta-casein grown in 42 L bioreactors. For details, see Example 4. A. Cells transformed with a vector expressing beta-casein were heated at 95°C for 30, 60, 90 or 120 min (30';60';90';120'). Lysates were centrifuged and soluble and insoluble fractions were analyzed by SDS-PAGE. S: Supernatant; P: Pellet. B. Cells transformed with a vector expressing beta-casein were heated at 55°C for 30, 60, 90 or 120 min (30';60';90';120') or at 95°C for 10, 20, 30, 60, 90 or 120 min (10;20;30';60';90';120'). The lysates were centrifuged and both soluble and insoluble fractions were analyzed by SDS-PAGE. 0': non-heated sample; C: sample incubated at room temperature for 120 min. [Diagram 5] Monitoring of partially purified beta-casein produced in E. coli by SDS-PAGE. (A) Samples were prepared as described in Example 5 by harvesting and resuspending cells, heating at 95°C for 90 min and further processing by centrifugation and filtration. (B) To compare the resuspended cell pellet (lysate) before and after heating, samples were prepared as described in Example 5. Filtered samples were also monitored. W: wash; C: resuspended cell pellet; H: lysate after heating; S: lysate supernatant after centrifugation; P: lysate pellet; F: final filtered sample. [Figure 6] Production of curds from partially purified beta-casein. A partially purified preparation of beta-casein as described in Example 5 was heat dried and mixed with other ingredients as described in Example 6. The resulting mixture was mixed with lemon, moulded and incubated to form curds (A). A mixture made with the same ingredients except for casein. This control mixture partially solidified on cooling but collapsed as observed and had poor water retention (B). [Figure 7]Purification of beta-casein by pyrolysis and precipitation in acidic conditions. Samples were treated and analyzed as described in Example 7. A. Monitoring of beta-casein by SDS-PAGE. Beta-casein was heated at 40°C or 90°C for 20 min and centrifuged at 3220g for 20 min after heat lysis at 95°C for 2 h, isolation and filtration of the supernatant (S) and adjustment of the pH of this supernatant to pH=4. After this final centrifugation step, both the supernatant and the pellet were analyzed: supernatant after heating at 40°C (S40), pellet after heating at 40°C (P40), supernatant after heating at 90°C (S90), pellet after heating at 90°C (P90). B. Monitoring of recombinant DNA in resuspended cell pellets (CP) at various steps of the purification process, including: in the supernatant after 2 h heating at 95°C (lane A), after adjusting the pH of this supernatant to pH 4 (lane B), after further heating at 90°C for 20 min, centrifugation and resuspension of the precipitated casein (C), after washing and resuspension of the precipitated casein (D), and after addition of Ca(OH)2 (Ca). Control PCRs were made in the absence of DNA or cell samples (-), or in the presence of a recombinant plasmid expressing beta-casein (+). M: 1 kb + NEB marker. [Figure 8] Coagulation with calcium caseinate and gelling agent. LpCC was seeded in the presence or absence of gelling agent (agar). Composition and protocol are explained in the examples. At the end of the draining step the preparation is made. Left: Coagulation in the absence of gelling agent. Center: Coagulation in the presence of 0.68% agar. Right: Agar 0.68%, no coagulant. [Figure 9] Coagulation with calcium caseinate and addition of gelling agent after coagulation. Composition and protocol are described in Example 6. Preparations are made after the end of the draining step. Left: 0.53% agar added after coagulation. Right: 0.53% agar added in the absence of coagulation. [Figure 10]Coagulation of recombinant caseins. Composition and protocol are described in Example 10. Left: Coagulation with beta-casein. Coagulation was performed in the presence of lipids and carbohydrates, but in the absence of gelling agents. At the end of the draining step the preparation is produced. Right: Coagulation of alpha-S1 and beta-caseins at acidic pH. No lipids or carbohydrates were added to the casein composition. Left and right pictures are not to scale. [Figure 11] Production of soft cheese from recombinant casein. Composition and protocol are described in Example 11. The preparation is produced at the end of the draining process. Left: Coagulation of beta-casein (batch 1) in the presence of 0.68% agar. Center: Coagulation of beta-casein, 0.68% agar, no coagulant. Right: Coagulation of alpha-S1 and beta-casein in the presence of 0.68% agar. [Figure 12] Addition of gelling agent after coagulation of beta-casein. Composition and protocol are described in Example 11. Preparation is made after the end of the draining step. Left: 0.53% agar is added after coagulation. Right: 0.53% agar is added in the absence of coagulation. [Figure 13] Soft cheese replica made from recombinant beta-casein. The product was made as described in Example 12. The product is after 7 days of aging. [Figure 14] SDS-PAGE analysis of alpha-S1 and beta-casein at various steps of the purification process. Samples were treated and analyzed as described in Example 13. A: M: molecular weight marker; lane 1: lysate; lanes 2 and 4: clarified lysate, lane 3: insoluble pellet; lane 4: clarified lysate after treatment with activated charcoal. M: molecular weight marker. Band sizes are indicated to the left of the gel. [Figure 15] Cheese replicas. Cheese replicas were made as described in Example 14. The photo was taken 6 days after setting. Description of the Invention

[0030] In the context of the present invention, "casein" is any casein protein or mixture of casein proteins. Thus, "casein" is alpha-S1 casein, alpha-S2 casein, beta casein or kappa casein. In some cases, it may refer to any mixture of such proteins. The term "casein" may be used to describe casein proteins in general.

[0031] In the context of the present invention, the term "between" includes the upper and lower limits.

[0032] In the context of the present invention, the term "cheese replica" refers to a food product that has the essential characteristics of cheese in terms of nutritional value, appearance, texture and taste.

[0033] The term "fresh cheese" refers to cheese that, on a fat-free basis, has a moisture content of more than 80% (ratio of water to total product mass without fat) and a protein content of 2% to 15% by total weight (ratio of protein mass to total product mass).

[0034] The term "soft cheese" or "semi-soft cheese" refers to cheeses that have a moisture content (ratio of water to total product weight without fat) of 62% to 80% on a fat-free basis and a protein content (ratio of protein weight to total product weight) of 15% to 30% by total weight. Soft cheeses have a moisture content of 67% to 80% on a fat-free basis and semi-soft cheeses have a moisture content of 62% to 67% on a fat-free basis.

[0035] In the context of the present invention, the term "liquid pre-curd composition" or "LpCC" refers to a composition containing at least casein and at least one other ingredient, including at least one of the following components: water, calcium, lipid, carbohydrate, prior to the addition of rennet and ferment and coagulation (particularly coagulation of casein). The casein concentration of LpCC is higher than that of milk. The concentrations of other ingredients in LpCC are also higher than those in milk. This allows some water to be saved when using LpCC with recombinant casein as disclosed in this application, since LpCC is not an artificial (cow) milk. The respective concentrations of proteins, lipids, calcium salts and / or carbohydrates are adjusted to suit the final desired composition of the cheese replica.

[0036] In the context of the present invention, the term "coagulant" refers to a chemical or biochemical composition capable of causing coagulation. Coagulation can be achieved by the addition of an acid solution, by the addition of a starter culture (a ferment whose growth in the presence of carbohydrates causes a drop in pH), by heat treatment, by the addition of a calcium chelating agent, by the addition of natural or recombinant rennet, by the addition of rennet substitutes such as animal proteases or vegetable clotting enzymes), or by a combination of these processes. The coagulant may be any additive, compound, composition or treatment that, alone or in combination with another or other additive, compound, composition or treatment, results in coagulation upon addition. When preparing animal-free edible compositions, it is preferred to use an acidifying agent (such as an acid or a starter culture) so that the coagulant does not contain elements of animal origin, in order to avoid the use of rennet. It is advantageous if the acidifying agent comprises one or more lactic acid bacteria, but acidic chemicals can also be used.

[0037] In the context of the present invention, the term "curd" means a composition in which casein has coagulated or precipitated under the action of a coagulating agent and which can be separated from a liquid phase, if one is present, for example by draining on cheesecloth. The curd also contains other components such as water and lipids, if present.

[0038] In the context of the present invention, the term "ferment" refers to a composition containing at least one microbial strain that is added during the manufacturing process of the cheese replica. Lactic acid ferment causes lactic acid fermentation, which in particular results in the acidification of the medium. As a result, the lactic acid ferment can be used as a coagulant.

[0039] Other ferments are used in cheese making to process the curd, resulting in changes in texture, taste, aroma and chemical composition (particularly the cleavage of proteins into smaller peptides). These ferments are sometimes called "maturation ferments", "ripening ferments" or "ripening ferments" and are not generally used in the production of fresh cheese. Such maturation ferments can be added together with the coagulating agent.

[0040] In the context of the present invention, the term "non-animal origin" refers to a compound or composition that is not derived directly from an animal, is not produced from an animal cell in culture, or is not isolated from an animal product such as milk. Thus, a compound or composition produced by fermentation of a microorganism is of "non-animal origin", even if products of animal origin, such as bactopeptone, may be involved during fermentation. Thus, in the context of the present invention, a protein that is naturally produced in an animal is called of non-animal origin when it is produced in a microbial (such as bacterial or yeast) cell or a plant cell, even if its sequence or structure is identical to that of a protein isolated from an animal.

[0041] In the context of the present invention, the term "animal-free" refers to a compound or composition that is not derived from animals, not derived from animal cells in culture, or not derived from animal products such as milk, and whose production process does not include any ingredients or additives of animal origin.

[0042] In the context of the present invention, the term "texturizing agent" refers to 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 agar (or agar-agar), carrageenan, tragacanth gum, xanthan gum, carob (locust bean) gum, cellulose gum, etc.

[0043] In a first aspect, there is provided a method for producing a casein composition comprising the steps of: i) providing a composition comprising casein and other proteins; ii) heating the composition so as to reduce (or decrease) the amount of other proteins in the soluble fraction of the composition; iii) recovering the soluble fraction; thereby obtaining a casein composition.

[0044] In particular, in ii), heating increases the ratio of casein to other proteins in the soluble fraction, thus enriching the composition for casein.

[0045] The composition of i) contains a soluble fraction and optionally an insoluble fraction. The soluble fraction refers to the fraction that is not pelleted by centrifugation. The insoluble fraction is the pellet obtained after centrifugation. Centrifugation is preferably carried out at about 3000 g for 20-30 minutes. On an industrial scale, the soluble fraction may be recovered using continuous flow centrifugation, or another method, such as filtration.

[0046] The desired technical effect is obtained by heating, preferably in the absence of, or without the addition of, an organic solvent.

[0047] Increasing the relative amount of casein compared to the total amount of protein increases the proportion of casein in the soluble fraction.

[0048] Such methods can be used to isolate or purify casein from compositions containing casein and other proteins, and can also be used to enrich casein in a protein composition, i.e., to increase the proportion of casein in the protein fraction of a protein composition.

[0049] In the context of the methods described herein, a "casein composition" refers to a composition that contains casein. To use a purification process based on the separation of soluble and insoluble fractions, the casein composition must contain a soluble fraction. However, the casein composition can then be dried in a further step. In some embodiments, casein comprises more than 25% of the composition's protein. In other embodiments, casein comprises more than 50% of the composition's protein. In some come embodiments, casein comprises the majority (more than 50%) of the dry weight. Such casein compositions can also contain other compounds, particularly calcium, other proteins, lipids, etc. Casein compositions produced by microbial culture are casein compositions of non-animal origin.

[0050] 1. A method for concentrating casein in a composition containing casein and other proteins, comprising the steps of: i) providing a composition containing casein and other proteins; ii) heating the composition so as to increase or maintain the amount of casein in the soluble fraction while reducing the amount of other proteins in the soluble fraction of the composition; iii) recovering the soluble fraction; thereby concentrating casein in the composition.

[0051] The other proteins are non-casein proteins. This method therefore makes it possible to obtain a composition enriched in casein. Enrichment of casein in a composition refers to the increase in the proportion (or relative amount) of casein in the total protein content, as compared to the amount before heating.

[0052] The desired technical effect is obtained by heating, preferably in the absence of, or without the addition of, an organic solvent.

[0053] With this in mind, it follows that the casein composition may contain only casein at the end of the process, or may also contain other proteins (if the precipitation and / or degradation of other proteins by heating is not complete). However, as indicated above, it is preferred if casein represents at least 50% (w / w) of the protein in the composition obtained after carrying out the process. In preferred embodiments, casein represents at least 60%, or at least 70%, or at least 80%, or at least 85%, or at least 90%, or at least 91%, or at least 92%, or at least 93%, or at least 94%, or at least 95% of the protein in the composition obtained after carrying out the process. The amount of protein can be measured by any method known in the art (use of gels, administration of protein).

[0054] As indicated above, the casein- and other protein-containing composition used in (i) may also contain other components such as carbohydrates or lipids. This is particularly true when the casein- and other protein-containing composition is obtained from the culture of a recombinant microorganism (including a eukaryotic cell) expressing casein. Such other components (in whole or in part) will be present in the casein composition.

[0055] Such compositions containing casein and other proteins for use in (i) may contain the microorganism suspended in a suitable buffer or solution, but more preferably in water, or, where casein is secreted by the microorganism, the supernatant obtained from a culture of the microorganism.

[0056] In certain embodiments, the starting composition (a composition containing casein and other proteins) contains alpha-S1 casein, hi one embodiment, alpha-S1 casein is the only casein in the starting composition.

[0057] In certain embodiments, the starting composition (a composition containing casein and other proteins) contains alpha-S2 casein, hi one embodiment, alpha-S2 casein is the only casein in the starting composition.

[0058] In certain embodiments, the starting composition (a composition containing casein and other proteins) contains beta-casein, hi one embodiment, beta-casein is the only casein in the starting composition.

[0059] In certain, but not preferred, embodiments, the starting composition (the composition containing casein and other proteins) contains kappa-casein. Thus, in preferred embodiments, the starting composition (the composition containing casein and other proteins) does not contain kappa-casein.

[0060] In certain embodiments, the starting composition (a composition containing casein and other proteins) contains a mixture of alpha-S1 casein and beta casein, hi one embodiment, alpha-S1 casein and beta casein are the only caseins in the starting composition.

[0061] In certain embodiments, the starting composition (a composition containing casein and other proteins) contains a mixture of alpha-S1 and alpha-S2 casein, hi one embodiment, alpha-S1 and alpha-S2 casein are the only caseins in the starting composition.

[0062] In certain embodiments, the starting composition (a composition containing casein and other proteins) contains a mixture of alpha-S2 casein and beta casein, hi one embodiment, alpha-S2 casein and beta casein are the only caseins in the starting composition.

[0063] In certain embodiments, the starting composition (a composition containing casein and other proteins) contains a mixture of alpha-S1 casein, alpha-S2 casein and beta casein, hi one embodiment, alpha-S1 casein, alpha-S2 casein and beta casein are the only caseins in the starting composition.

[0064] 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.

[0065] In certain embodiments, the starting composition (a composition containing casein and other proteins) contains a mixture of a combination of two to three caseins selected from alpha-S1 casein, alpha-S2 casein, beta casein and kappa casein.

[0066] The soluble fraction may be subjected to further purification steps such as filtration (in particular ultrafiltration, nanofiltration, reverse osmosis), chromatography or further precipitation such as acidic precipitation of casein in order to further purify and / or concentrate the casein. In particular, the acidic precipitation is carried out at pH=4 and preferably at about 90° C. Several other purification steps can be carried out. Also, activated carbon can be added to the soluble fraction before or after carrying out one or more of the above purification methods.

[0067] In a particular embodiment, the microorganism is a bacterial cell. In another embodiment, the microorganism is a fungal cell (including a yeast cell). In another embodiment, the microorganism is a eukaryotic cell, in particular a plant cell. Indeed, it is preferred that casein is produced in a non-animal organism.

[0068] 2. A method for producing a casein composition taking advantage of the property of casein (remaining soluble even at high temperatures while other proteins precipitate and / or degrade) when produced by the culture or fermentation of a microorganism (particularly a bacterium) transformed to express casein, comprising: i) providing a microbial composition (preferably a bacterial composition), the microbial composition (preferably a bacterial composition) comprising a microorganism (preferably a bacterium) transformed with at least one nucleic acid encoding casein, the microorganism (preferably the bacterium) being cultured so as to express and produce casein; ii) heating the microbial composition (preferably a bacterial composition) at a temperature that induces enrichment of casein in the soluble fraction and / or a reduction in the amount of other proteins in the soluble fraction; iii) isolating a soluble fraction from the heated solution of ii); iv) optionally carrying out a further step selected from the addition of activated carbon applied to the soluble fraction isolated in iii), membrane filtration (ultrafiltration, nanofiltration or reverse osmosis), chromatography or precipitation of casein; thereby obtaining a casein composition.

[0069] In ii), heating the microbial composition (preferably a bacterial composition) also induces lysis of cells, if they are not already lysed, to produce a microbial extract, enrich the soluble fraction for casein and / or reduce the amount of other proteins in the soluble fraction.

[0070] Thus, if the microbial composition contains already lysed microorganisms, the heating in ii) will concentrate the casein in the soluble fraction and / or reduce the amount of other proteins in the soluble fraction, and a further effect of lysis of the microorganisms will be observed if the microorganisms present in the microbial composition are not already lysed.

[0071] An enrichment of casein or a reduction in other proteins is observed in the soluble fraction compared to the soluble fraction of a microbial composition in which the microorganisms are lysed prior to heating.

[0072] Thus, such a method allows obtaining a casein composition and isolating a casein-rich (or casein-enriched) composition from a microbial culture. A casein-rich composition is a composition in which casein is the major protein. In a preferred embodiment, casein represents more than 50% of the protein in said casein-rich composition. In a more preferred embodiment, casein represents more than 80%, or 90% or 95% of the protein in said casein-rich composition.

[0073] Indeed, as shown in the examples, upon heating, casein moves from the insoluble fraction (pellet) to the soluble fraction (supernatant), and the examples also show that it is essentially casein that moves to the soluble fraction under the action of heat.

[0074] The microbial composition contains microbial cells suspended in a suitable fluid (e.g., water or other buffer as described below). It is therefore possible to obtain an insoluble (pellet) fraction and a soluble fraction (supernatant) by centrifugation. However, this is preferably performed before centrifugation. In this embodiment, the insoluble fraction may be suspended in the soluble fraction when heating is performed. Thus, centrifugation or another method (such as filtration) may be used to recover the soluble fraction.

[0075] In one embodiment, the microbial composition (preferably a bacterial composition) is obtained by centrifugation of the microbial cells (preferably bacteria) after culturing, washing, and resuspension in a suitable liquid or fluid (in a suitable buffer, and more preferably in water, more preferably in the absence of organic solvents). The buffer has a pH that is not acidic (above 6.5), preferably close to neutral (6.5-7.5) or basic (preferably below 9). Indeed, casein may precipitate at acidic pH, and this process is further enhanced by heating, as shown in Example 7. This is also preferred if the buffer is not ionic or has a low ionic strength. The soluble fraction in iii) can be obtained by centrifugation of the heated composition in ii) or by other methods known in the art.

[0076] The composition of i) in the method disclosed above is heated in ii) at a temperature of about 75°C to about 100°C, in particular 75°C to 105°C. Such a temperature makes it possible to obtain the technical and functional effects described in ii) (removal of other proteins and therefore reducing their amount and / or transferring casein from the insoluble fraction to the soluble fraction). When using water, it is also possible to function at temperatures above 100°C by increasing the pressure. The skilled person is able to determine the appropriate conditions of temperature, pressure and pH for carrying out the method disclosed herein.

[0077] The term "about," when referring to a measurable value, is meant to encompass a ±4% variation from the specified value, such variation being appropriate for performing the disclosed methods.

[0078] In particular, the temperature is 80° C. or higher, or 85° C. or higher, or 90° C. or higher. In particular, the temperature is about 85° C. to about 95° C., more particularly about 95° C. In another embodiment, the temperature is about 90° C. to about 95° C. The temperature is generally below 100° C. Temperatures of 80° C. to 100° C., or 85° C. to 100° C., are preferred.

[0079] The duration of heating can be adjusted by those skilled in the art. When heating is performed at about 95°C, the duration may be from about 5 minutes to about 2 hours. It is preferable that the duration is at least 10 minutes, 20 minutes, 30 minutes, 40 minutes, 1 hour, and more preferably about 2 hours. The duration can be extended (especially at low temperatures or particularly at high temperatures, it can be shortened).

[0080] As indicated above, certain embodiments encompass the situation where the starting composition (a composition containing casein and other proteins) is obtained from a bacterial culture, in such embodiments, the bacteria has been transformed with one or more nucleic acids encoding two or more caseins.

[0081] In particular, in one embodiment the bacterium is transformed with one or more nucleic acids encoding beta-casein.

[0082] In particular, in one embodiment the bacterium is transformed with one or more nucleic acids encoding alpha-S1 casein.

[0083] In particular, in one embodiment the bacterium is transformed with one or more nucleic acids encoding alpha-S2 casein.

[0084] In particular, in one embodiment the bacterium is transformed with one or more nucleic acids encoding both beta-casein and alpha-S1 casein.

[0085] In particular, in one embodiment the bacterium is transformed with one or more nucleic acids encoding beta-casein, alpha-S1 casein and alpha-S2 casein, or a combination of two of them.

[0086] It should be noted that if multiple proteins are produced, the bacteria can be transformed with different nucleic acids (each encoding a different protein) or with a unique nucleic acid (containing elements that allow for the production of the various proteins). Such methods for transforming microorganisms and bacteria, particularly for the production of one or more proteins, are known in the art.

[0087] The nucleic acids are such that they contain all the elements (promoter, enhancer, terminator, etc.) that allow the production of the casein they code for. The skilled artisan knows all these elements and is able to select the most appropriate ones. The skilled artisan is also able to select the microorganism to be used to produce the casein.

[0088] In particular, a suitable prokaryotic host for the expression of casein is Escherichia coli ( E. coli ), Bacillus subtilis ( Bacillus subtilis ), Salmonella Typhimurium ( Salmonella typhimurium ), Lactococcus lactis ( Lactococcus lactis ), and Lactococcus ( Lactococcus ) genus, Pseudomonas ( Pseudomonas ) genus, Streptomyces ( Streptomyces ) and Staphylococcus ( Staphylococcus ) genus.

[0089] Suitable eukaryotic hosts for the expression of casein include Saccharomyces cerevisiae ( Saccharomyces cerevisiae ), Kluyveromyces lactis ( Kluyveromyces lactis ), Pichia pastoris ( Pichia pastoris ), or Trichoderma reesei ( Trichoderma reesei ) and other fungi.

[0090] Plant cells can also be used to produce proteins, either in cell culture or in the whole plant.

[0091] Further steps such as the addition of activated carbon, membrane filtration, chromatography or precipitation of casein can be added, resulting in i) providing a microbial composition (preferably a bacterial composition), the microbial composition (preferably a bacterial composition) comprising a microorganism (preferably a bacterium) transformed with at least one nucleic acid encoding casein, the microorganism (preferably the bacterium) being cultured so as to express and produce casein; ii) heating the microbial composition (preferably a bacterial composition) at a temperature that induces enrichment of casein in the soluble fraction and / or a reduction in the amount of other proteins in the soluble fraction; iii) isolating a soluble fraction from the heated composition of ii); iv) further processing the soluble fraction by adding at least one step selected from the addition of activated carbon, membrane filtration, chromatography or casein precipitation; thereby obtaining a casein composition.

[0092] In this embodiment, the heating in ii) also induces lysis of the microorganisms, thereby obtaining a microbial extract in which casein is enriched and / or other proteins are reduced.

[0093] Furthermore, the heating process is separate from the cell lysis. i) providing a microbial extract, the microbial extract resulting from lysis of a microbial culture comprising a microorganism transformed with a nucleic acid encoding casein and cultured to express and produce casein; ii) heating the microbial extract at a temperature to concentrate casein in the soluble fraction and / or reduce the amount of other proteins in the soluble fraction of the composition; iii) isolating the soluble fraction after heating; iv) further processing the soluble fraction by adding at least one step selected from the addition of activated carbon, membrane filtration, chromatography or casein precipitation; and thereby obtaining a casein composition.

[0094] The soluble fraction isolated in iii) may be further processed to increase the purity of the casein.

[0095] Activated carbon can be added to the soluble fraction and stirring is performed. Activated carbon can be used to adsorb impurities (particularly organic impurities or chlorine) that are present in the soluble fraction and that were not removed during recovery of the soluble fraction in iii). Stirring with activated carbon is preferred and the activated carbon is removed before performing another process on the soluble fraction, such as the process disclosed below.

[0096] Chromatographic techniques are widely used purification methods and include affinity chromatography, ion exchange chromatography, hydrophobic interaction chromatography, among others.

[0097] Membrane filtration, especially ultrafiltration and nanofiltration, is also 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, they can be used to separate different caseins from each other (see above).

[0098] Finally, 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 its tendency to precipitate under acidic conditions can be exploited to carry out further purification.

[0099] In a preferred embodiment, further processing of the soluble fraction is carried out by precipitation of the caseins in acidic conditions. After precipitation, the caseins can be resuspended and resolubilized using an appropriate 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). Precipitation in acidic conditions is particularly interesting when isolating caseins from microbial cultures, since it allows the removal or degradation of any nucleic acids that may be present in the casein composition. When using caseins to obtain edible compositions (such as cheese replicas) intended for human consumption, the presence of microbial DNA or RNA may prove to be harmful (at least from a regulatory point of view).

[0100] In certain embodiments, the casein composition is dried, and when obtained from a bacterial culture, such compositions contain about 15-30% casein (w / w) and carbohydrates in the dry composition.

[0101] It is preferred that a small amount of water remains to facilitate future hydration. Thus, drying should be understood as reducing the amount of water. In some embodiments, the amount of water is about 50% or less (w / w).

[0102] In a particular embodiment, the present invention provides a method for producing a casein composition comprising the steps of: i) providing a bacterial composition, the bacterial composition comprising a bacterium transformed with at least one nucleic acid encoding casein, the bacterium being cultured to express and produce casein; ii) heating the bacterial composition at a temperature that induces enrichment of casein in the soluble fraction and / or a reduction in the amount of other proteins in the soluble fraction; iii) isolating a soluble fraction from the heated cell composition of ii); iv) further processing the soluble fraction by adding at least one step selected from the addition of activated carbon, membrane filtration, chromatography or casein precipitation; thereby obtaining a casein composition.

[0103] If the bacteria of the bacterial composition are not lysed, the heating in ii) also induces lysis of the cells and produces a bacterial extract.

[0104] In another embodiment, the present invention provides a method for producing a casein composition comprising the steps of: i) providing a bacterial composition, the bacterial composition comprising a bacterium transformed with at least one nucleic acid encoding casein, the bacterium being cultured to express and produce casein; ii) heating the bacterial composition at a temperature that induces enrichment of casein in the soluble fraction and / or a reduction in the amount of other proteins in the soluble fraction; iii) isolating a soluble fraction from the heated bacterial composition of ii); iv) further processing the soluble fraction by precipitation of casein under acidic conditions; thereby obtaining a casein composition.

[0105] In this embodiment, if the bacteria of the bacterial composition are not lysed, the heating in ii) shall also induce lysis of the bacteria, thereby producing a bacterial extract.

[0106] In another embodiment, the present invention provides a method for producing a casein composition comprising the steps of: i) providing a bacterial composition, the bacterial composition comprising a bacterium transformed with at least one nucleic acid encoding casein, the bacterium being cultured to express and produce casein; ii) heating the bacterial composition at a temperature that induces enrichment of casein in the soluble fraction and / or a reduction in the amount of other proteins in the soluble fraction; iii) isolating a soluble fraction from the heated bacterial composition of ii); iv) further treating the soluble fraction by precipitation of casein by heating at 90° C. at pH=4; thereby obtaining a casein composition.

[0107] In a preferred embodiment, cell lysis is induced by heating the bacterial composition.

[0108] The process disclosed herein therefore makes it possible to obtain a casein composition. Such a casein composition obtainable (or obtainable) by the process disclosed herein is a further subject of the present invention. Such a composition can be characterized as defined above.

[0109] In milk, casein is in a micellar form found in milk, which includes alpha-S1, alpha-S2, beta, and kappa caseins assembled in the same particle. In the context of the present invention, the casein does not have to be assembled in a micellar form. However, as shown below, this does not preclude the ability to obtain curds from the casein composition by adding a suitable coagulant.

[0110] Indeed, as detailed in the examples, such casein compositions can be used to produce cheese replicas, and in particular animal-free cheese replicas, i.e. cheese replicas that do not contain any products of animal origin (especially when the casein is isolated from bacterial or yeast cultures).

[0111] In summary, by simply heating a composition containing casein and other proteins, the casein in the composition can be concentrated by reducing the amount of most of the other proteins in the soluble fraction. Heating can also solubilize the casein found in the insoluble fraction of bacterial cultures. The resulting casein is not in an optimal state to form micelles, but can be properly coagulated by the action of a coagulant. Such a discovery was unexpected.

[0112] In particular, it has been shown that recombinant casein produced in bacteria can be used to achieve coagulation in the absence of kappa-casein.

[0113] It is known that dairy caseins, especially alpha-S1, alpha-S2 and beta caseins, are phosphorylated and furthermore kappa casein is glycosylated (Walstra et al. (2006) Dairy Science and Technology. Taylor and Francis Group, Boca Ranton, USA; Martin et al. (2003) Non bovine caseins quantitative variability and molecular diversity. In: Fox PF and Mc Sweeney PLH. Advances in Dairy Chemistry-Proteins, Vol1, Springer, New York, 227-310). Casein phosphorylation plays an important role in the interaction with calcium and the structure of dairy casein micelles. Such post-translational modifications are not found in recombinant caseins produced in bacteria.

[0114] It is also known that kappa casein plays an important role in the formation of dairy casein micelles. WO 2020 / 223700 describes the formation of micelles using recombinant alpha-S1 casein (dephosphorylated) and kappa casein (deglycosylated), followed by the production of milk curds using these two caseins (see Example 14 of the document). However, WO 2020 / 223700 also reports that the use of low phosphorylated alpha casein results in looser micelles (see Example 11 of the document). Therefore, the fact that coagulation can be achieved in the absence of post-translational modifications and kappa casein is unexpected.

[0115] The composition disclosed herein and obtained or obtainable by the method disclosed above can therefore be used in a method for obtaining curdled milk, the method comprising the steps of: i) providing a casein composition as disclosed herein; ii) mixing said casein composition with at least one other ingredient comprising at least one component selected from the group consisting of water, calcium, lipids and carbohydrates to obtain a Liquid Pre-curd Composition (LpCC); iii) adding at least one coagulant to said liquid composition to obtain curd; Includes.

[0116] In a preferred embodiment, the coagulation agent is an acidifier, not rennet. In a preferred embodiment, the coagulation agent is a lactic acid bacteria, or lactic, citric or acetic acid. In a preferred embodiment, the casein composition does not contain any kappa casein. In a preferred embodiment, the casein does not contain any post-translational modifications. In one embodiment, the casein composition contains only alpha-S1 casein (as the casein proteins). In one embodiment, the casein composition contains only alpha-S2 casein (as the casein proteins). In one embodiment, the casein composition contains only beta casein (as the casein proteins). In one embodiment, the casein composition contains only alpha-S1 casein and beta casein (as the casein proteins). In one embodiment, the casein composition contains only alpha-S2 casein and beta casein (as the casein proteins). In one embodiment, the casein composition contains only alpha-S1 casein and alpha-S2 casein (as the casein proteins). In one embodiment, the casein composition contains only alpha-S1 casein and alpha-S2 casein (as the casein proteins). In one embodiment, the casein composition contains alpha-S1 casein, alpha-S2 casein and beta casein.

[0117] The present invention also provides a method for producing an edible composition, comprising the steps of: i) providing a casein composition as disclosed herein; ii) mixing said casein composition with at least one other ingredient comprising at least one component selected from the group consisting of water, calcium, lipids and carbohydrates to obtain a Liquid Pre-curd Composition (LpCC); iii) adding at least one coagulant to the liquid composition to obtain curd; iv) further processing the curd to obtain an edible composition; The present invention relates to a method comprising the steps of:

[0118] In a preferred embodiment, the coagulation agent is an acidifying agent rather than rennet. In a preferred embodiment, the coagulation agent is a lactobacillus or lactic acid, citric acid or acetic acid.

[0119] In the above embodiment, it is preferable to add a gelling agent in step b) or d).

[0120] It is preferred if all other ingredients added in b) are of non-animal origin. In the process for producing the curd and edible compositions disclosed herein, it is preferred to use only products of non-animal origin, so as to obtain an edible composition that does not contain elements of animal origin.

[0121] In a preferred embodiment, the component added in b) is i. optionally, a protein other than casein; ii. lipids, iii. Water, and iv. Carbohydrates in a preferred embodiment, the carbohydrates are lactose-free to make the edible composition more acceptable to lactose-intolerant customers.

[0122] Generally, the edible composition comprises: i. protein content is less than 25%, preferably 2-25% or 5-25% (w / w); ii. The moisture content is greater than 62% on a fat-free basis.

[0123] In a preferred embodiment, the edible composition is a cheese replica.

[0124] In a preferred embodiment, the casein composition contains at least one casein selected from the group consisting of alpha-S1-, alpha-S2-, beta-, or kappa-casein. In a more preferred embodiment, the casein composition contains at least two caseins selected from the group consisting of alpha-S1-, alpha-S2-, beta-, or kappa-casein. In a more preferred embodiment, the casein composition contains alpha-S1-, alpha-S2-, beta-, or kappa-casein. In one embodiment, at least one casein selected from the group consisting of alpha-S1 casein, alpha-S2 casein, beta casein, and kappa casein is absent from the casein composition. In one embodiment, kappa casein is absent from the casein composition. In one embodiment, the casein composition contains only beta casein. In one embodiment, the casein composition contains only beta and alpha-S2 caseins. In one embodiment, the casein composition contains only beta and alpha-S1 caseins. In one embodiment the casein composition contains only beta, alpha-S1 and alpha-S2 caseins. Indeed, since the inventors have shown that it is possible to obtain edible compositions that are curds and cheese replicas without the use of kappa casein, the inventors have not attempted to reconstitute micelle formation or to confirm whether the solution actually exhibits micelles.

[0125] When preparing an animal-free edible composition, it is preferred to use an acidifying agent (such as an acid or a starter culture) so that the coagulating agent does not contain elements of animal origin, in order to avoid the use of rennet. It is advantageous if the acidifying agent comprises one or more lactic acid bacteria.

[0126] The composition of the LpCC is adapted to the final composition desired for the edible composition, taking into account water loss due to coagulation and aging, which can be adjusted by one skilled in the art by varying the duration and conditions of maturation.

[0127] In one embodiment, the cheese replica has the essential characteristics of fresh cheese. Fresh cheese contains 80% or more moisture on a fat-free basis. The percentage of moisture on a fat-free basis is the mass of water present in the product divided by the total mass minus the mass of fat (= mass of water / total mass-mass of fat). The protein content of fresh cheese is usually less than 15%, and may be 7% or even lower of the total weight.

[0128] In this embodiment, the edible composition preferably comprises: i. The protein content is 2-15% (by weight); ii. The moisture content is greater than 80% on a fat-free basis.

[0129] More specifically, the edible composition will have a protein content of 5 to 15% (weight content).

[0130] The LpCC obtained in b) is i. protein content is less than 12% (by weight); ii. Moisture content is greater than 80% on a fat-free basis In some cases, these features can be obtained.

[0131] In particular, the LpCC obtained in b) is i. The protein content is 5% to 10% (weight content); ii. The moisture content is greater than 80% on a fat-free basis.

[0132] In this embodiment, it is preferable to add agar to LpCC in an amount equivalent to 0.5% to 0.8% (w / w) of LpCC.

[0133] Some other features of the LpCC used in the edible composition or in the process for making the edible composition are described below. These features can be found alone or in the composition: the lipid content of the edible composition is between 10% and 30% (weight content); - The lipid content of LpCC is 10% to 30% (weight content); the calcium content of the edible composition is less than 2.8% (by weight); The calcium content of LpCC is 0.1% to 1% (weight content).

[0134] When carrying out the process to obtain an edible composition that will be a fresh cheese replica, the treatment of the curds includes: a) mixing a structurant with the curd to obtain a supplemented curd, the structurant being selected from the group consisting of gelling agents, texturizing agents, emulsifiers and mixtures thereof; b) forming and draining the replenished curd; may include: c) Other embodiments are also disclosed below.

[0135] In this embodiment, the invention provides a method for producing an edible composition, comprising: a) providing a casein composition comprising a recombinant casein as disclosed herein (preferably only recombinant casein as disclosed herein, preferably only casein of non-animal origin); b) mixing said casein composition with at least one other ingredient comprising at least one component selected from the group consisting of water, calcium, lipids and carbohydrates to obtain a Liquid Pre-curd Composition (LpCC); c) adding at least one coagulant to the liquid pre-curd composition to obtain a curd; d) further processing the curd to obtain an edible composition; Including, A gelling agent is added in b) and / or d).

[0136] "Containing only casein of non-animal origin" indicates that there is no casein of animal origin (i.e., containing only casein of non-animal origin). In a preferred embodiment, other elements, if present, are also of non-animal origin. In another embodiment, the casein composition contains no elements other than casein (of non-animal origin, preferably recombinant casein as described herein).

[0137] In one embodiment, a gelling agent is added in b).

[0138] In another embodiment, the gelling agent is added in d).

[0139] In another embodiment, the gelling agent is added in b) and d).

[0140] In this embodiment, the edible composition i. The protein content is 2-15% (by weight); ii. Moisture content is greater than 80% on a fat-free basis; This is preferable.

[0141] This embodiment is therefore well suited for producing fresh cheese replicas.

[0142] In another preferred embodiment, the cheese replica has the essential characteristics of a soft or semi-soft cheese. Soft cheeses contain 67-80% moisture on a fat-free basis and may be mould or washed type. Their protein content is often in the range of 20% of the total weight. Mould soft cheeses have a thick and creamy texture and the cheese has a slight elasticity. The aging process varies according to their thickness. The cheese has a mixed curd inoculated with a specific mould and slowly drained. Washed soft cheeses have a thick and creamy texture and the cheese has a slight elasticity. During the aging process, the cheese is periodically turned and brushed or washed with a brine with added beer, mead, wine or spirits. Semi-soft cheeses contain 62%-67% moisture on a fat-free basis. The texture may be soft and creamy. When aged, the cheese can be washed with a brine with a red smear (with or without alcohol) (washed type). The cheese may be brushed and / or allowed to develop a natural rind.

[0143] In particular, in this embodiment, the edible composition comprises: i. The protein content is 15-25% (by weight); ii. The moisture content is between 62% and 80% on a fat-free basis.

[0144] This is especially true for the LpCC obtained in b). i. protein content is 10-18% (weight content); ii. Moisture content is greater than 62% on a fat-free basis; This can be achieved in some cases.

[0145] In this embodiment, it is preferable to add agar to LpCC in an amount equivalent to 0.5% to 1% (w / w) of LpCC.

[0146] In this embodiment, it is advantageous if in c) a maturation ferment is added (ripening ferment) and d) involves the salting and shaping of the curd.

[0147] In particular, step d) comprises maturing the curd at a temperature allowing the development of a maturing ferment, in particular at 14° C. The maturation period is determined by the skilled artisan depending on the desired characteristics of the edible composition. The maturation period can be several days or several weeks.

[0148] Some other features of the LpCC used in the edible composition or in the process for making the edible composition are described below. These features can be found alone or in the composition: The lipid content of the edible composition is 10 to 30% (weight content), - The lipid content of LpCC is 10% to 30% (weight content); the calcium content of the edible composition is less than 2.8% (by weight); The calcium content of LpCC is 0.3 to 1.7% (weight content).

[0149] In one particular embodiment, the present invention provides a method for producing an edible composition, comprising the steps of: a) providing a casein composition comprising a recombinant casein as disclosed herein (preferably only recombinant casein as disclosed herein, preferably only casein of non-animal origin); b) mixing said casein composition with at least one other ingredient comprising at least one component selected from the group consisting of water, calcium, lipids and carbohydrates to obtain a Liquid Pre-curd Composition (LpCC); c) adding at least one coagulant to the liquid pre-curd composition to obtain a curd; d) further processing the curd to obtain an edible composition; Including, A gelling agent is added in b), a maturation ferment is added in c), and d) involves salting and shaping the curd.

[0150] In this embodiment, the edible composition i. The protein content is 15-25% (by weight); ii. Moisture content is between 62% and 80% on a fat-free basis This is preferable.

[0151] This embodiment is well suited for producing soft cheese replicas.

[0152] Furthermore, the present invention also provides i) providing a bacterial composition, the bacterial composition comprising a bacterium transformed with at least one nucleic acid encoding casein, the bacterium being cultured to express and produce casein; ii) heating the bacteria at a temperature that induces a concentration of casein in the soluble fraction and / or a reduction in the amount of other proteins in the soluble fraction; iii) isolating the soluble fraction after heating; iv) optionally further processing said soluble fraction 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 said casein composition with at least one other ingredient comprising at least one component selected from the group consisting of water, calcium, lipids and carbohydrates to obtain a Liquid Pre-curd Composition (LpCC); vi) adding at least one coagulant to the liquid composition to obtain curd; The present invention relates to a process including the steps of:

[0153] If the bacteria are not lysed, the heating in ii) is carried out at a temperature that induces lysis of the cells, thereby producing a bacterial extract.

[0154] In a preferred embodiment, the coagulation agent is an acidifying agent rather than rennet. In a preferred embodiment, the coagulation agent is a lactobacillus or lactic acid, citric acid or acetic acid.

[0155] The term "Liquid Pre-curd Composition" or "LpCC" refers to a composition containing at least casein and at least one other ingredient including at least one of the following components: water, calcium, lipid, carbohydrate, prior to the addition of rennet and ferment and coagulation (particularly coagulation of casein).

[0156] "Coagulant" refers to a chemical or biochemical composition capable of causing coagulation. Coagulation can be achieved by the addition of an acidic solution, by the addition of a starter culture (whose growth in the presence of carbohydrates causes a drop in pH), by the addition of natural or recombinant rennet, by the addition of rennet substitutes such as animal proteases or vegetable clotting enzymes, or by a combination of these processes. It is also possible to carry out a heat treatment or add calcium chelators, especially to improve or accelerate coagulation.

[0157] The coagulation agent may be any additive, compound, composition or treatment that, alone or in combination with another or other additive, compound, composition or treatment, results in coagulation upon addition.

[0158] The overall protein content (including casein and derived peptides) varies depending on the cheese type and production process. The protein content in the LpCC affects the protein content of the final product and can be adjusted to produce a desired type of cheese substitute. The protein content (weight content) in the LpCC is equal to the mass of protein in the LpCC divided by the total mass of the LpCC.

[0159] In a preferred embodiment, the protein content in the LpCC is less than 12% (weight content). In a more preferred embodiment, the content is between 2% and 10% (weight content). In a more preferred embodiment, the content is between 5% and 10% (weight content). These amounts are of particular interest for the production of fresh cheese substitutes.

[0160] In another preferred embodiment, the protein content in the LpCC is between 10% and 25% (weight content). In a more preferred embodiment, the content is between 10% and 18% (weight content). These amounts are particularly interesting for the production of soft or semi-soft cheese substitutes.

[0161] Calcium and other salts Milk contains about 1.2 g of calcium per liter (about 0.12% by weight). Calcium is present in cheese and contributes to its nutritional value. Calcium ranges from 0.1 to 1% of the cheese weight, with the lowest calcium contents usually found in fresh cheeses (0.125% by weight in the French "Fromage Blanc"), while the highest contents are usually found in hard cheeses (0.97% in Emmental). In Camembert, the calcium content is in the range of 0.25% by weight. During the cheese-making process, the amount of calcium remaining in the milk varies depending on the process (Mietton et Chablain, Du lait au fromage: les fondamentaux technologiques. In Gillis JC, Ayerbe A, Le fromage 4th edition, Lavoisier-Technique Et Documentation 20 avril 2018), with higher retention being achieved by using rennet (calcium 62-67%) than lactic acid coagulants (calcium 16-17%). Furthermore, the percentage of calcium in the LpCC will be important to closely reproduce the characteristics of a given cheese category. However, cheeses with an increased calcium content are also envisaged.

[0162] In a preferred embodiment, the calcium content in LpCC is 0.1% to 1.7%.

[0163] Some food additives containing calcium, especially calcium carbonate (E170), are of animal origin. However, there are also calcium sources of mineral or vegetable origin (such as lithothamne sea weed). The method disclosed herein offers the possibility to replace the calcium of milk with calcium of non-animal origin, thereby reducing the animal-origin constituents. In a more preferred embodiment, the calcium is of non-animal origin. Other salts such as phosphates, which are important constituents of milk, or others, can be added.

[0164] Lipids Lipids are present in most cheeses and affect the organoleptic sensation. However, in the last decades, cheeses with reduced fat content (low-fat) or even fat-free cheeses have been produced to address health concerns. The method disclosed herein offers the possibility to replace milk lipids with lipids of non-animal origin, thereby reducing the animal-origin components. In a more preferred embodiment, the lipids are lipids extracted from plants.

[0165] In cheese, the fat content varies from 0% to over 30% in completely fat-free products. The fat content in cheese is equal to the mass of fat in the cheese divided by the total mass of the cheese.

[0166] The fat composition may be adjusted in quantity and quality to mimic the composition of existing cheeses. Lipids are present in large amounts in most cheeses. 95%-10% of the fats in milk remain in the curds during coagulation. However, the lipid content varies greatly depending on the origin of the milk (cow, goat, sheep) and the preparation of the milk and the cheese making process.

[0167] The fat content can be as low as 7% or even lower in fresh cheeses, or over 30% in traditional hard cheeses. Modern industrial cheeses include products with very low fat content or very high fat levels (e.g., 36% of the total weight in Bongrain's Boursault, France) to meet the demand for fat-free products.

[0168] In the context of the present invention, the fat composition in the LpCC can be varied at will to obtain fat-free or fat-rich products. The fat content (weight content) in the LpCC corresponds to the mass of lipids in the LpCC divided by the total mass of the LpCC.

[0169] In a preferred embodiment, the fat composition in the LpCC is 0% (weight content). In another preferred embodiment, the fat composition is less than 10% (weight content). In another preferred embodiment, the fat composition is 10%-20% (weight content). In another preferred embodiment, the fat composition is 20%-30% (weight content). In another preferred embodiment, the fat composition is 30%-40% (weight content).

[0170] The lipids in milk consist essentially of triglycerides, molecules resulting from the fusion of a glycerol molecule with three fatty acids by esterification, but this composition changes significantly during cheese processing, with hydrolysis of the triglycerides producing various amounts of mono- and diglycerides and free fatty acids, which can be further processed and play a very important role in the taste and aroma of the final product.

[0171] The lipids may be included as a composition of lipids extracted from plants. The main fatty acids in milk fat are palmitic acid (31%), oleic acid (24%), myristic acid (12%), stearic acid (11%), smaller sized saturated acids (11%), palmitoleic acid (4%), linoleic acid (3%), trans-unsaturated acids (3%), and alpha-linoleic acid (1%). Such a composition can be reproduced in LpCC. However, the composition can also be adjusted, for example to increase the proportion of unsaturated fatty acids.

[0172] Dairy products are rich in saturated fatty acids, and in milk, saturated palmitic, myristic and stearic acids, together with smaller size molecules, account for 65% of fatty acids.One of the advantages of using lipid compositions, for example, from plants, is that they can provide a healthier fat source.Indeed, vegetable fats usually contain less saturated and trans fatty acids than animal fats.Furthermore, their composition can be enriched with lipids known to have health benefits, such as omega-3 fatty acids, including, for example, alpha-linolenic acid, eicosapentaenoic acid or docosahexaenoic acid.

[0173] In the context of the present invention, various lipid sources of plant origin can be used, such as canola or rapeseed, sunflower, soybean, coconut oil, olive oil, walnut oil, hazelnut oil, and margarine. Such products are mostly commercially available. However, products of sufficient purity should be used to avoid carryover of undesirable flavors and / or tastes. Deodorized oil can also be used. Deodorization is a steam stripping process in which good quality steam, generated from deaerated and properly treated feed water, is injected into soybean oil under low absolute pressure and high enough temperature to evaporate free fatty acids (FFA) and odorous compounds and carry these volatiles away from the feedstock. Various soybean oil processing processes are described in Practical Handbook of Soybean Processing and Utilization (1995), OCS Press. Elsevier Inc.

[0174] In a preferred embodiment, the lipid is present in canola oil, rapeseed oil, sunflower oil, soybean oil, palm oil, olive oil, walnut oil, hazelnut oil or margarine. In a more preferred embodiment, the lipid is present in canola oil, rapeseed oil, soybean oil or palm oil.

[0175] Emulsifiers and gelling agents An emulsifier or emulsifying agent is a compound or substance that acts as a stabilizer for an emulsion and prevents the separation of liquids that normally do not mix. It can be useful to use an emulsifier to avoid the formation of solid and liquid phases during and after the preparation of LpCC. Lecithin is very widespread. The term actually refers to a family of amphiphilic compounds found in animals and plants, but one can easily find soy lecithin or sunflower lecithin commercially available.

[0176] In a preferred embodiment, an emulsifier is added to avoid the formation of liquid and solid phases. In a more preferred embodiment, the emulsifier is lecithin. The emulsifier can be added to the LpCC in step b) or before (or during) the addition of the coagulating agent in c) (however, the emulsifier should be mixed with the LpCC before coagulation).

[0177] Alternatively, or in addition, gelling agents such as agar or guar gum, gum arabic, tragacanth gum, xanthan gum, carob (locust bean) gum, cellulose gum, cassia gum, sesbania gum, tamarind gum, fenugreek gum, carrageenan, etc. may be used. Such hydrocolloids add firmness and improve water retention. Gelling agents of animal origin, such as gelatin, should be excluded from the composition.

[0178] The addition of a gelling agent (mixed with the curd) before or after setting is of particular interest in the context of the present invention. This will help to give texture to the edible composition. In particular, locust bean gum can be used.

[0179] Of particular interest is agar (added to LpCC in an amount equivalent to 0.5%-0.8% (w / w) of LpCC, or 0.5%-1% (w / w) of LpCC).

[0180] carbohydrates In another preferred embodiment, the other components contain at least calcium, lipids and carbohydrates. Lactose is the main carbohydrate of milk. Cow's milk contains 70 mg / L lactose, 20 mg / L galactose, and trace amounts of other carbohydrates, including various oligosaccharides. During cheese ripening and in ripened cheese, carbohydrates are eventually completely or almost completely decomposed. However, they are still present in significant amounts in fresh cheese, which should usually be avoided by people with strong lactose intolerance. In any case, the individual reaction to each type of cheese varies depending on the severity of lactose intolerance.

[0181] Lactose is important because it is the raw material for the microflora (ferment) of cheese. However, it can be replaced by glucose or other carbohydrates. Carbohydrates such as glucose, saccharose or fructose are of plant origin, but the majority of lactose available on the market today is of animal origin. The method disclosed herein offers the possibility to replace milk lactose with glucose or another carbohydrate of non-animal origin, thereby reducing the animal origin components, which ultimately provides the additional benefit of addressing lactose intolerance.

[0182] In a preferred embodiment, no lactose is added to the composition. In a more preferred embodiment, a carbohydrate of non-animal origin is added to the composition. In an even more preferred embodiment, the carbohydrate is glucose, saccharose or fructose.

[0183] vitamin In a more preferred embodiment, the other ingredients include at least calcium, lipids, carbohydrates and vitamins. Dairy products are usually rich in vitamins. Milk contains water-soluble vitamin B 12Milk is a good source of vitamins B1, B2, B8, B5, but contributes little to the need for vitamin B3. Milk also provides the fat-soluble vitamin A, but is a poor source of vitamins D, E, and K. However, in cheeses, which are usually concentrated in fat, this low fat-soluble vitamin content is mitigated. Cheese substitutes may have the same nutritional value in terms of vitamins as traditional cheeses, or may be supplemented with higher amounts of vitamins. However, in the context of the need for alternatives of non-animal origin, which may often be associated with a vegan diet or a diet with a low content of animal products, the vitamin B 12 is usually the most important additive to be provided, since it is essentially provided by food of animal origin. In a more preferred embodiment, the composition is supplemented with at least one vitamin. In an even more preferred embodiment, this vitamin is vitamin B 12 It is.

[0184] In a more preferred embodiment, the other ingredients include at least calcium, lipids, carbohydrates and vitamin B12.

[0185] Coagulant Milk clotting can be achieved by the addition of acid or simply by lactic acid fermentation, but most cheeses today are made using rennet, most often in conjunction with lactic acid bacteria. Digestion of kappa-casein by rennet enzymes destabilizes the casein / calcium phosphate micelles, causing coagulation and resulting in the formation of curds. Rennet will therefore be used in most cases. However, as explained above, there are various types of rennet. Traditional rennet is obtained from calf stomachs, but other types of rennet have also been developed, including vegetable rennet and rennet derived from recombinant microorganisms. Such non-animal rennets can be purchased, for example, from Chr. Hansen Lab. (DK), Gist Brocades / DSM. (NL), Pfizer Inc. (USA) (see Le fromage Gillis JC, Ayerbe A (2018), Lavoisier ed. Paris).

[0186] In a more preferred embodiment, a coagulant of non-animal origin is added. In a preferred embodiment, the coagulant is an acidifier, not rennet. In a preferred embodiment, the coagulant is a lactic acid bacterium, or lactic acid, citric acid or acetic acid.

[0187] Fermentation In a more preferred embodiment, a ferment is added. Lactic acid bacteria are an essential component of cheese ferments. They contribute to lactic acid production, which then regulates the pH, thereby modifying the physicochemical conditions and, together with rennet, contributes to the texture of the final product. In an even more preferred embodiment, the ferment comprises at least one lactic acid bacteria. In an even more preferred embodiment, the lactic acid bacteria is Streptococcus cremoris ( Streptococcus cremoris ), Streptococcus lactis ( Streptococcus lactis ), Streptococcus diacetylactis ( Streptococcus diacetylactis ), Streptococcus thermophilus ( Streptococcus thermophilus ), Leuconostoc lactis ( Leuconostoc lactis ) , Leuconostoc citrovorum ), Lactobacillus bulgaricus ( Lactobacillus bulgaricus ), and Lactobacillus helveticus ( Lactobacillus helveticus ) is selected.

[0188] Lactic acid bacteria are the first microorganisms to spread in milk and curds, modifying the composition of the curds and paving the way for the next group of ferments. Together with this next group of ferments, lactic acid bacteria, including non-starter lactic acid bacteria (NSLAB) and other microorganisms, contribute to maturation or aging. In an even more preferred embodiment, the ferment comprises at least one NSLAB. In an even more preferred embodiment, this NSLAB is Lactobacillus paracasei ( Lactobacillus paracasei ) , Lactobacillus casei ), Lactobacillus rhamnosus ( Lactobacillus ramnosus ), Lactobacillus plantarum ( Lactobacillus plantarum ) , Lactobacillus brevis ), Lactobacillus buchneri ( Lactobacillus bucheneri , Lactobacillus fermentum ( Lactobacillus fermentum ) , Enterococcus faecalis ), Enterococcus faecium ( Enterocuccus faecium ), Pediococcus pentosaceus ( Pediococcus pentosaceus ), and Carnobiobacterium larutalomatium ( Carbiobacterium laltaromatium ).

[0189] In an even more preferred embodiment, the ferment contains at least another microorganism which is not a lactic acid bacterium, but may be a yeast, a mold or a non-lactic acid bacterium. Debaryomyces hansenii ), Geotritium Candidium ( Geotrichum candidum ), Kluyveromyces larxianus ( Kluyveromyces larxianus ), Kluyveromyces lactis ( Kluyveromyces lactis ), Saccharomyces cerevisiae ( Saccharomyces cerevisiae ), Candida catenulatus ( Candida catenulate ), Candida intermediata ( Candida intermedia ), Torulaspora delbrueckii ( Torulaspora delbrueckii ), Candida zeylanoides (Candida zeylanoides ), Pichia membranifaciens ( Pichia mebranifaciens ), Candida rugiosa ( Candida rugiosa ), and Pichia fermentans ( Pichia fermentans ).

[0190] In an even more preferred embodiment, the fermentation is Penicillium camenbergii ( Penicillium camemberti ), Penicillium roqueforti ( Penicillium roqueforti ), Chrysosporium sulphureum ( Chrysosporum sulfureum ), Fusarium domesticum ( Fusarium domesticum ), Isomucor fuscus ( Isomucor fuscus ), Mucor plumbeus ( Mucor plumbeus ), Penicillium commune ( Penicillium commune ), and Splendenoma casei ( Sprendonema casei ).

[0191] Fermentates are commercially available from a number of companies including, for example, Chr. Hansen.

[0192] Water (moisture) In milk, water accounts for about 87.4% (by weight) and 90.8% on a fat-free basis. The water content (by weight) is the mass of water divided by the total weight in a given volume. The water content (called moisture) on a fat-free basis is the mass of water present in the product divided by the total mass minus the mass of fat (= mass of water / (total mass-mass of fat)). Moisture is an important characteristic of cheese varieties and is the main criterion for cheese varieties as defined below.

[0193] Ideally the moisture in the LpCC should be as low as possible to avoid waste whilst still allowing some drainage. For soft or semi-soft and firm or hard cheeses (see below) the moisture content of the LpCC can be significantly below that of milk.

[0194] In a preferred embodiment, the moisture content in the LpCC is less than 90% on a fat-free basis, in a more preferred embodiment, the moisture content is less than 80%.

[0195] When dry casein is used, water and fat as well as emulsifiers can be added to obtain LpCC. As an example, when dry casein containing about 23% casein, water and other elements (such as carbohydrates) is used, 1.3 g water, about 2 g fat and about 0.027 g emulsifier can be added for 1 g dry casein. The pH can be adjusted to about 5-5.5 using a suitable acidic agent (lemon juice can be used, but also lactic acid, citric acid or acetic acid).

[0196] The fat is preferably a vegetable fat such as peanut oil, coconut oil, canola oil, hazelnut oil, walnut oil, avocado oil, olive oil, etc. It is preferable to use a neutral oil (i.e. an oil without taste or odor). In particular, deodorized coconut oil can be used.

[0197] The emulsifier is selected from the list of such emulsifiers approved for food use, and may include lecithin (especially soy lecithin), propylene glycol fatty acid esters (PGMS), monoglycerides and diglycerides.

[0198] Processing of milk curds The curd can then be further processed to obtain a cheese replica.

[0199] The processing of such curds may involve one or more steps such as: - cutting and heating the curd, - draining the curds to remove the water so that the curds can form a mat (this corresponds to the removal of whey in traditional cheese processing using milk); - giving texture to the curd: the curd can be cut into pieces, stacked, flipped and pressed to allow more water to be expelled if necessary or to allow fermentation to continue; - drying and / or salting and / or brining the product, - forming a block of cheese replica Includes.

[0200] The substitutes may be stored and aged, particularly to allow for maturation and development of flavor and texture.

[0201] The processing of the curds is essentially similar to the processing of curds to make cheese. EXAMPLES

[0202] Example 1: Monitoring recombinant casein in soluble and insoluble fractions of E. coli extracts The native casein genes code for precursor proteins that contain a signal peptide. In mammals, this peptide is cleaved off during casein processing and is not present in the mature protein in milk. The synthetic genes encoding alpha-S1, alpha-S2 and beta-casein (related to the native genes P02662, P02663 and P02666, respectively) were modified to remove the signal peptide. The sequences of the new synthetic open reading frames are shown in the last column of Table 1). The genes were cloned into pET25b+ and the resulting plasmids were transformed into the BL21(DE3) strain. Individual transformed clones were isolated and one clone per synthetic gene was used to inoculate LB medium. Clones transformed with the empty vector (pET25b+) were used as controls.

[0203] [Table 2-1] [Table 2-2] [Table 2-3] [Table 2-4]

[0204] Cells were lysed using two different protocols and samples of total extracts and soluble and insoluble fractions were analyzed by SDS PAGE. Casein was monitored in the soluble and insoluble fractions of cell extracts using two different protocols. Cells transformed with empty vector (pET25b+) were used as a control.

[0205] Protocol 1: Cell pellets from 100 mL cultures were lysed by resuspension in lysis buffer (50 mM Tris HCl (pH 7.5), 1 mg / mL lysozyme, 0.03 mg / mL Dnase). The suspension was incubated on ice for 30 min and then sonicated for 10 s (10% amplitude, Q Sonica XL-2000). 10 μL of the total fraction was collected and stored for SDS-PAGE analysis. Soluble and insoluble fractions were separated by centrifugation at 3220 g for 20 min at 4° C. The supernatant was collected and supplemented with 10% glycerol for storage. The pellet was further resuspended in Tris 50 mM with 2% SDS and supplemented with 10% glycerol for storage. 10 μL of each fraction was taken for SDS-PAGE.

[0206] Protocol 2: Cell pellets from 100 mL cultures were lysed by resuspension in Bugbuster (Millipore) containing 0.4% Lysonase (Millipore). The mixture was incubated at room temperature for 5 min and then centrifuged at 3220 g for 20 min at 4° C. The supernatant was collected and 10 μL of the supernatant was taken and saved for SDS-PAGE analysis. The pellet was further resuspended in 2% SDS and 10 μL of the suspension, corresponding to the insoluble fraction, was taken and saved for SDS-PAGE analysis.

[0207] As shown in Figure 1, alpha-S1 casein was found in both the soluble (S) and insoluble (P) fractions (Figure 1A), whereas alpha-S2 was found essentially in the insoluble fraction (Figure 1B). As is often observed with overexpression of recombinant proteins in E. coli, most (Protocol 1) or all (Protocol 2) of the beta-casein was found in the insoluble fraction (Figure 1C). This suggests that in E. coli, casein may be present in inclusion bodies. The different quantitative results may be due to different solubilities of beta-casein or different stabilities of inclusion bodies depending on the protocol. Nevertheless, recovery of casein from the soluble fraction of the extract using these protocols results in loss of most or all of the recombinant protein.

[0208] Example 2: Monitoring recombinant casein in soluble and insoluble fractions of E. coli extracts resulting from heating The effect of heat lysis on casein was tested using the recombinant strains described in Example 1. A clone transformed with the empty vector (pET25b+) was used as a control.

[0209] The culture was centrifuged and the cell pellet was resuspended in 1 volume of sterile water, centrifuged, washed in another volume of water and resuspended in 1 volume of water. 1 mL samples of this cell suspension were treated by heating at 95°C for 0 (unheated), 10, 20, 30, 60, 90 and 120 min, resulting in cell lysis, and soluble and insoluble fractions were separated by centrifugation. The insoluble fraction was resuspended in 1 mL of buffer (50 mM Tris HCl (pH 7.5), 300 mM NaCl, 10 mM MgCl2, 2 mM DTT, 0.5% Triton and Sigmafast Protease Inhibitor (Sigma)) and 10 μL aliquots of both the soluble and insoluble fractions were analyzed by SDS-PAGE. Under these conditions, a 10 μL sample of the soluble fraction and a 10 μL sample of the insoluble fraction are approximately the same amount of total cell extract. When no heat was applied (0' in Figure 2), no cell lysis or only lysis of residual cells in water occurred. The pellet contained essentially whole cells, including casein, and this sample actually represents a whole cell extract.

[0210] Surprisingly, it was observed that temperature-induced dissolution influenced the distribution of casein in the soluble and insoluble fractions (Figure 2).

[0211] By (or before) 10 minutes of heating at 95°C, alpha-S1 casein was found almost entirely in the soluble fraction (Figure 2A), whereas in Example 1 it was found in similar amounts in both the soluble and non-soluble fractions.

[0212] Casein beta was gradually transferred to the soluble fraction containing the bulk of this recombinant protein by (or before) 10 minutes of heating, and was mostly transferred by 30 minutes (Figure 2C). In Example 1, beta-casein was found mostly or entirely in the insoluble fraction.

[0213] The distribution of casein alpha-S2 (which in Example 1 was entirely in the insoluble fraction) appeared to be less heat sensitive, and by 120 min of heating it was still found predominantly in the insoluble fraction (Figure 2B), nevertheless it appeared in the soluble fraction by 20 min of heating at 95°C, and decreased slightly in the insoluble fraction over time.

[0214] Many other protein bands from the insoluble fraction also decreased over time (Figures 2A, 2B, and 2C) but did not migrate to the soluble fraction. In addition to the casein bands, only a few faint protein bands were visible in the soluble fraction of the heated samples.

[0215] These results indicate that lysis by heat is a good method to carry out rapid purification of caseins, especially alpha-S1 and beta-casein.

[0216] Example 3: Production of recombinant beta-casein in a 42 L fermentor Cultures of the recombinant strains described in Example 1 were used to inoculate a 42 L fermentor (Biostat CPlus, Sartorius).

[0217] 30 L of LB medium supplemented with 30 g / L yeast extract (NuCel 751 MG) and 0.1 mg / L ampicillin was poured into a 42 L fermenter. Initial OD 600 The fermenter was inoculated with a preculture of the recombinant clone until the OD reached 0.06. The fermentation was carried out in fed-batch mode, feeding the culture with a solution containing 143 g / L D-glucose and 214 g / L yeast extract at pH=7, T=37°C, pO2=10%. Ampicillin was added after 24 hours (0.1 mg / L culture). After 17 hours, the culture reached an OD of approximately 10 and production was started by adding IPTG (1 mM final). The culture reached an OD of 41.3 at T=41.3, OD 600 The reaction was stopped at =44. The cells were harvested by centrifugation at 10°C.

[0218] For analysis, aliquots of cells were centrifuged and resuspended in one volume of lysis buffer (50 mM Tris HCl (pH 7.5), 300 mM NaCl, 10 mM MgCl2, 2 mM DTT, 0.5% Triton and Sigmafast Protease Inhibitor (Sigma)). The suspension was incubated on ice for 30 min, then sonicated for 1 min (15% amplitude, QSonica XL-2000) and 10 μL of all fractions were analyzed by SDS-PAGE (Figure 3A).

[0219] Beta-casein purified from milk (Sigma) was used as a control, but its apparent molecular weight was higher, as previously observed by others (Simons et al. Overproduction of bovine beta-casein in Escherichia coli and engineering of its main chymosin cleavage site (1993) Protein Engineering 7: 763-770). Recombinant casein production was estimated to be in the range of 3 g per L of bacterial culture.

[0220] Samples were also analyzed for the presence of recombinant beta-casein in the soluble and insoluble fractions using the same two protocols as in Example 1. The results were similar to those observed in Example 1, with most (protocol 1) or all (protocol 2) of the beta-casein (casein beta) found in the insoluble fraction (Figure 3B).

[0221] Example 4: Monitoring recombinant beta-casein in soluble and insoluble fractions of E. coli extracts resulting from heating at different temperatures The effect of heat lysis on recombinant beta-casein was tested in cultures of the strains described in Example 3.

[0222] One liter of cells (approximately 15 g dry cell weight) was centrifuged, the cells were resuspended in 1 liter of sterile water, centrifuged, washed with another liter of water, and resuspended in 1 liter of water. 70 mL samples of this cell suspension were treated by heating at 95°C for 30, 60, 90, and 120 minutes, resulting in cell lysis, and soluble and insoluble fractions were separated by centrifugation. The insoluble fraction was resuspended in 1 volume of lysis buffer (50 mM Tris HCl (pH 7.5), 300 mM NaCl, 10 mM MgCl2, 2 mM DTT, 0.5% Triton, and Sigmafast Protease Inhibitor (Sigma)) and a 10 μL aliquot was analyzed by SDS-PAGE.

[0223] As shown in FIG. 4A, consistent with the results observed in Example 2 with heat lysis, and in contrast to the results observed in Example 1 with the other heat lysis protocol, the overexpressed protein was found predominantly in the soluble fraction by (or before) 30 minutes of heating.

[0224] Many other protein bands from the insoluble fraction also decreased over time but did not migrate to the soluble fraction. In addition to the beta-casein band, some faint protein bands were visible in the soluble fraction of the heated samples, but decreased over time, an effect that was barely observable in Example 2, possibly due to the lower sample concentration.

[0225] These results are consistent with the progressive thermal degradation of lysate proteins observed in studies dedicated to the production and purification of thermophilic or thermostable proteins (Takesawa et al. (1990) Heat-induced precipitation of cell homogenates: an investigation of the recovery of thermostable proteins. Enzyme Microb.Technol.12, 184-189; Kirk and Cowan 1995 Optimising the recovery of recombinant thermostable proteins expressed in mesophilic hosts. J. of Biotechnology 42: 177-184; Sundarrajan et al. (2018) Novel properties of recombinant Sso7d-Taq DNA polymerase purified using aqueous two-phase extraction: Utilities of the enzyme in viral diagnosis. Biotechnol Rep (Amst) 19: e00270; US Pat. No. 8,603,782; WO 2011 / 119703).

[0226] Following the same protocol, the effect of various temperatures, namely 55°C, 75°C and 95°C, on both the soluble and insoluble fractions was also tested. As shown in Figure 4B, at 55°C, a slight increase in the amount of beta-casein in the soluble fraction could be observed by 120 min. At 75°C, a decrease in other soluble proteins in the soluble (and insoluble) fractions was observed by 1 h, with a more pronounced recovery of beta-casein in the soluble fraction. At 95°C, beta-casein was clearly observed in the soluble fraction by 10 min of heating, while other bands decreased over time in both the insoluble and soluble fractions, with this last observation becoming evident in both fractions by 30 min of heating.

[0227] These results showed that heat-induced lysis is interesting for the rapid purification of beta-casein, both by solubilizing intracellular caseins and by removing non-casein proteins.

[0228] It has also been shown that heating can be used to separate casein from other non-heat stable proteins, separate from dissolution.

[0229] Example 5: Biopurification of Recombinant Beta-Casein A protocol was established for the biopurification of casein beta from cells.

[0230] Cell samples equivalent to 15 g dry cell weight were resuspended in 1 L of sterile water. Cells were treated by heating at 95° C. for 90 min. The lysate was centrifuged at 3220 g for 20 min at 4° C., and the supernatant was filtered through a 0.2 μm membrane using a vacuum-driven filtration system (Stericup Quick Release, Millipore).

[0231] Aliquots of samples from the various steps were analyzed by SDS-PAGE. As shown in Figure 5, the final filtered protein fraction (lane F) was heavily enriched in recombinant protein, which ultimately accounted for the majority of protein in the preparation.

[0232] Moreover, protocols involving heat lysis can have very high yields. In another experiment, cell samples equivalent to 7.5 g dry cell weight were washed, resuspended in sterile water, and monitored for beta-casein by SDS-PAGE before and after heating at 95 °C for 90 min. The amount of beta-casein was found to be very similar in these two conditions and also in the filtered samples of the heated extract (Figure 5B). The amount of beta-casein was estimated by visual comparison with a standard protein preparation, showing that approximately 3 g of recombinant protein was observed per liter of culture in all three samples. Similar quantitative results were observed when samples from the same culture were heated at 100 °C and 2 bar pressure for 30 min instead of 95 °C for 90 min.

[0233] Example 6: Preparation of milk curds from partially purified beta-casein The casein solution (batch 1) described in Example 5 was dried by heat. Casein in this extract was quantified using SDS-PAGE analysis, and it was estimated that 23% of the dried product represented beta-casein.

[0234] 1.8 g of the dried casein preparation (0.41 g casein) was mixed with 2.4 g water and incubated at room temperature for 3.5 hours to allow complete rehydration. 3.5 g deodorized coconut oil (BioPlanete, France) and 0.05 g lecithin were added and the pH was adjusted to 5 with 0.1 g lemon juice. The mixture was incubated at 4° C. for 30 minutes for molding and then demolded at room temperature.

[0235] The final composition (excluding chives) is summarized in Table 2 (composition 1) and the product characteristics are shown in Figure 6A. Upon demoulding, it exhibits a firm and stable texture at room temperature with perfect water retention. The casein content is estimated to be 5.3%.

[0236] This texture and water retention was not achieved without the addition of 1.8 g of the dried casein preparation (composition 2 in Table 2, FIG. 6B). Instead, a solid phase due to the coconut oil was observed, but it crumbled upon removal from the mold and significant water loss was observed.

[0237] [Table 3]

[0238] Example 7: Purification of recombinant beta-casein by heat and acid precipitation and monitoring of recombinant DNA A sample of the culture described in Example 3 was treated as described in Example 5. The cells were resuspended in sterile water, centrifuged, resuspended in water, the suspension was heated at 95°C for 2 hours, the lysate supernatant was isolated and filtered through a 0.2 μm membrane. The supernatant was then heated under acidic conditions to precipitate casein. The pH of the supernatant was lowered to pH 4 with 0.1 M HCl solution and then heated at 40°C or 90°C for 20 minutes. The suspension was then centrifuged at 3220g for 20 minutes and the supernatant and pellet were analyzed by SDS-PAGE. As shown in Figure 7A, heating under such acidic conditions partially precipitated casein beta at 40°C and completely at 90°C.

[0239] To achieve a neutral pH solution, an additional step was added to this protocol: the pellet containing precipitated casein was washed once with H2SO4 solution (pH 4) and once with sterile water, resuspended in sterile H2O, and the pH was adjusted to 7 with 1 M Ca(OH)2 to yield a calcium caseinate suspension (not shown).

[0240] Heating in acidic conditions is known to accelerate DNA degradation. To assess the presence of DNA from microbial producing strains in casein preparations, the same protocol was applied to another sample (however, the filtration step was skipped) and the DNA was monitored using PCR amplification. The reaction was performed using two primers designed to amplify a 929 bp region containing the beta-casein coding sequence.

[0241] [ka]

[0242] [ka]

[0243] PCR reactions were performed with 1 μL of sample in 20 μL using DreamTaq Green PCR Master Mix (2X) (ThermoScientific). PCR was performed using a SimpliAmp thermal cycler (Applied Biosystems) with cycling conditions as follows: 95°C for 5 min, 30 cycles of main reaction (95°C for 30 s, 61.4°C for 30 s, 72°C for 1 min), and 72°C for 5 min. Amplification products were loaded onto a 1% agarose gel and visualized using a GeneFlash (Syngene) UV transilluminator. As shown in Figure 7B, recombinant DNA was still detectable under these conditions after pyrolysis (lane A) and after adjusting the pH of the supernatant to pH 4 (lane B), but was not detectable after heating at 90°C under such acidic conditions (lane C) and in subsequent steps (lane D and Ca).

[0244] Example 8: Testing the effect of gelling agents on the preparation of fresh cheese replicas Mixture of calcium caseinate, agar, water, deodorized coconut oil and glucose: 18 g of calcium caseinate casein (Armor Proteine, containing 92% casein as caseinate, 1% lipid and 1% calcium) were resuspended in 195 g of water to obtain a 7.8% (w / w) casein in water suspension. 15 grams of this casein composition were mixed with 2 g of deodorized palm oil (BioPlanete, France) and 2.5 g of a 25% (w / w) glucose solution in water. The new composition was heated to 45° C. and 4 ml of a heated 0%, 2%, 3% or 4% agar preparation (agar dissolved in water) was added with gentle mixing. Samples 1 to 4 were replicated as samples 5 to 8 (samples 1, 4 and 8 were replicated in a second independent experiment). The resulting composition (LpCC) is listed in Table 4. The pH is approximately 7.0.

[0245] [Table 4]

[0246] coagulation 0.2 g of lactic acid ferment (Streptococcus thermophilus, Lactobacillus bulgaricus, Alsa) was added to samples 1-4 (initial temperature 25°C) but not to samples 5-8 (see Table 5), and the compositions were incubated at 40°C for 10 hours. In samples 1-4, the pH dropped to about 4.5, indicating that lactic acid fermentation was actively taking place. In samples 5-8, the pH remained stable at about 7.0.

[0247] Drainer The entire composition was gently stirred to break up the smooth structure obtained with the highest agar concentration, placed on cheesecloth, and drained at 4°C for 16 hours. The curd weights ranged from about 7 g to about 15 g, as reported in Table 5. The compositions were estimated (Table 5) based on the assumption that the casein, lipids, and agar were all retained in the curd. In fact, very little color was observed in the drained liquid of Samples 1-4, and no palm oil or agar coagulation was observed in the drained liquid at 4°C. In contrast, the estimates for calcium and glucose are maximum estimates, and some of these compounds may be found in the drained liquid. For the same reason, the estimates for moisture content (on a total and non-fat basis) are minimum estimates.

[0248] [Table 5]

[0249] The results show that the gelling agent can be used to adjust the moisture and therefore the overall composition: with 0.68% agar, over 60% of the LpCC was retained (versus 30% without gelling agent), and the product had the expected casein content and moisture of fresh cheese, as well as a plastic texture and appearance (Figure 8).

[0250] In the absence of lactic acid fermentation, samples 5–8 obtained solid phases of various sizes depending on the agar concentration. The coloration of the draining liquid suggests that some of the casein was lost from the solid phase, so estimates are not shown in Table 4. The solid phases obtained without the use of fermentants were smaller compared to their fermented curd counterparts (Table 4), but also retained a loose structure (Figure 8).

[0251] When gelling agent is added after solidification The same effect was achieved by adding a gelling agent after coagulation: Sample: LpCC with similar composition was incubated with lactic acid bacteria at 40°C for 10 hours. The pH dropped from about 7.0 to about 4.5. After coagulation, 3 ml of 4% agar in water as above was added and the entire mixture containing the curd and liquid phase was gently stirred and placed on cheesecloth for draining.

[0252] The compositions before coagulation (LpCC), after coagulation and addition of agar, and after draining are estimated in Table 6. The composition after coagulation and draining is specified as above. After 16 hours of draining at 4°C, approximately 12 g of curd was obtained in two independent samples, showing better water retention (53% of total) than that observed in the absence of gelling agent (see above).

[0253] This product had the expected casein content and moisture of fresh cheese (Table 6), and plastic texture and appearance (Figure 9). Thus, for certain types of cheese that remain in a semi-solid form, it can be added either before or after coagulation.

[0254] This procedure was reproduced in the absence of fermentation. A lighter solid phase (approximately 6 g, 27% of the total) was obtained. However, the texture was very loose, as shown in Figure 9.

[0255] [Table 6]

[0256] Example 9: Production of recombinant alpha and beta casein batches The native casein gene encodes a precursor protein that includes a signal peptide. In mammals, this peptide is cleaved during casein processing and is not present in the mature protein in milk. As shown in Example 1, synthetic genes encoding alpha-S1 and beta-casein (related to the native genes P02662 and P02666, respectively) were modified to remove the signal peptide. The sequences of the new synthetic open reading frames are shown in the last column of Table 7).

[0257] [Table 7]

[0258] The resulting ORF was overexpressed in bacteria and casein was purified according to the method described in the previous examples. Finally, three batches of animal-free recombinant casein were obtained as calcium caseinate. To obtain concentrated calcium caseinate, the casein extract precipitated with H2SO4 at pH=4.6 was resuspended in water and the pH was slowly adjusted to 7 with Ca(OH)2. The preparation was concentrated by evaporation using a Rotavapor R-300 (Buchi) device.

[0259] Batch 1: Beta-casein (calcium caseinate), 150 mg / g composition, pH 7. Batch 2: Alpha-S1 casein and beta casein (calcium caseinate), approximately 50 mg / g and 25 mg / g respectively, pH=7. Batch 3: Alpha-S1 casein and beta casein (calcium caseinate), approximately 6 mg / g and 3 mg / g respectively, pH=7.

[0260] Although other proteins and other organic molecules were probably present in these batches, casein was estimated to represent over 90% of the total protein and over two-thirds of the dry weight.

[0261] Example 10: Clotting studies of partially purified recombinant beta-casein A casein composition containing recombinant beta-casein was used to test the clotting ability of recombinant beta-casein in the absence of a texturizing agent using batch 1 from Example 9. For this, a preparation was prepared with approximately the same casein, lipid and water content as sample 1 in Example 8, but recombinant beta-casein was used instead of commercial caseinate.

[0262] The batch 1 preparation was diluted with water to obtain a recombinant beta-casein composition with a concentration of 58.7 mg / g. 20 g of this casein composition was mixed with coconut oil, glucose and agar dissolved in water to obtain 23.5 g of a composition containing 5% casein, 8.5% lipid, 2.7% glucose and 0.68% agar in water. Additional carbohydrates and proteins may come from the casein composition as well as salts, but are not considered in this table. Thus, the moisture content is likewise only an estimate, but the error in the moisture content should not exceed 3%, considering that in the initial casein composition casein made up at least 2 / 3 of the dry weight.

[0263] The composition was inoculated with 0.3 g of lactic acid ferment and incubated at 40° C. for 10 hours. The pH dropped from about 7.0 to about 4.5. The entire composition was then placed on cheesecloth and drained at 4° C. for 16 hours. After draining, 7 g of curd could be obtained, although it was not as hard as that obtained under similar conditions using commercial caseinate (FIG. 10).

[0264] The ability of alpha-S1 and beta-casein from Example 7 to coagulate efficiently at low pH was simply tested by adding lactic acid to 90 ml of low concentration Batch 3 composition to adjust the pH to about 4.5. After separation by filtration on cheesecloth, about 0.7 g of material was obtained (Figure 6). In contrast, when no lactic acid was added to the same volume of composition, the entire composition flowed through the cheesecloth.

[0265] Example 11: Production of fresh cheese replicas from partially purified recombinant casein The batch 1 preparation was diluted with water to obtain a recombinant beta-casein composition at a concentration of 78.2 mg / g. 15 g of this casein composition was mixed with coconut oil, glucose and agar as described in Example 8 to obtain the composition described in Table 5. Additional carbohydrates and proteins may come from the casein composition as well as salts, but are not considered in this table. Thus, the moisture content is likewise only an estimate, but should not be more than 3% error, given that casein made up at least 2 / 3 of the dry weight in the initial casein composition.

[0266] The composition was inoculated with 0.3 g of lactic acid ferment and incubated for 10 hours at 40° C. The pH dropped from about 7.0 to about 4.5. The entire composition was then gently stirred, placed on cheesecloth, and drained at 4° C. for 16 hours.

[0267] The curd weight was about 14 g. Its composition (Table 8) was estimated according to the same rules as above and in Example 9. About 60% of LpCC was retained. The product obtained had the necessary casein content and moisture for fresh cheese. The texture was not as hard as the commercial calcium caseinate of Example 8, but it was still suitable for fresh cheese and showed plasticity and hardness to be molded into a stable shape (Figure 11). For comparison, a sample without lactic acid bacteria was prepared according to the same procedure. Without the use of a coagulant, a solid phase of about 13 g could be obtained, but the structure was loose and could not stably hold the shape (Figure 11).

[0268] The same procedure was followed using batch 2 of casein containing alpha-S1 casein and beta casein. Although only 38% of the LpCC was retained, likely due to the low protein content (2.5% of LpCC), the resulting product had the casein content and moisture desired for fresh cheese. The texture was not as firm as the commercial calcium caseinate of Example 8, but still suitable for fresh cheese, exhibiting plasticity and firmness for molding into stable shapes (Figure 11).

[0269] [Table 8]

[0270] When gelling agent is added after solidification Following the same procedure as in Example 8, the addition of gelling agent after coagulation was also tested. The compositions before coagulation (LpCC), after coagulation and addition of agar, and after draining are estimated in Table 9. The composition after coagulation and draining is the same as above. After lactic acid fermentation, addition of agar (0.53%), and draining in cheesecloth for 16 hours at 4° C., about 10 g of curd was obtained (44% of the total). Without the use of ferment, a lighter solid phase was obtained (about 6 g, 27% of the total). However, the texture was very loose, as shown in Figure 12.

[0271] [Table 9]

[0272] Example 12: Producing soft cheese replicas from partially purified recombinant casein The batch 1 preparation was further concentrated to reach a concentration of 250 mg / g of the composition.

[0273] 15g sunflower oil, 34g hydrated cashew nuts (for the composition of hydrated cashew nuts, see Example 5 above), 70g water and 1.5g agar were mixed. The mixture was brought to a boil and boiled for 10-20 seconds. 100g concentrated casein composition containing 25g recombinant beta-casein was added to the mixture and mixed gently. The LpCC composition details are shown in Table 10.

[0274] Coagulation, draining, salting and maturation 200 g of this LpCC (out of a total of 220.5 g) were inoculated with a ferment containing lactic acid bacteria (MBT, SOGEBUL, Dole, France), yeast (DH2d SOGEBUL, Dole, France) and an additional mature ferment (PC12H, SOGEBUL, Dole, France) at a temperature below 35° C. The composition was incubated in the mould at 20° C. for 24 hours.

[0275] The product is removed from the mold and placed on a draining grid. The product and grid are placed in a box to control humidity. Depending on the desired taste, about 1 / 4 to 1 / 2 teaspoon of table salt (La Baleine, France) can be spread on the top surface and the product is incubated at 14 °C. After 24 hours, the product is inverted and the same salt composition is spread on the opposite surface. To control humidity, the product is incubated on the grid in a purification box at 14 °C, inverted every 2 days for several weeks. During this period, water is periodically removed from the purification box. The product on the 7th day is shown in Figure 13.

[0276] The weight loss is essentially due to water. As the initial composition is known (the composition of dried cashew nuts is approximately 22% carbohydrate, 20% protein, 53% lipids, and a few percent water), the assessment of water loss can be used to calculate the product composition, especially in terms of moisture (62% to 80% moisture on a fat-free basis), or to achieve the expected composition or soft cheese. By the seventh day, the product was estimated to weigh 150 g, with protein and lipid contents of 17.4% (including 15.1% casein and derived peptides) and 16.6%, respectively, and moisture of 76% on a fat-free basis.

[0277] [Table 10]

[0278] Example 13: Purification of recombinant alpha-S1 and beta-casein A vector was constructed to co-express recombinant alpha-S1 and beta-casein. Essentially, both ORFs encoding alpha-S1 and beta-casein from Table 1 (P02662 and P02666) were inserted into pET25+, with beta-casein proximal to the promoter and a T7 ribosome binding site inserted in front of each of the two ORFs. The resulting plasmid was transformed into strain BL21(DE3). Individual transformed clones were isolated and one clone was used to inoculate LB medium. Bacterial growth and casein expression were then carried out in 42 L fermenters (Biostat CPlus, Sartorius) as shown in Example 3.

[0279] The final OD at the end of the fermentation was about 70. The cells were harvested by centrifugation (2000g, 30 min, +4°C) and stored at -80°C. A clear yellow supernatant with an OD between 0 and 1 was obtained. After thawing, the cells were resuspended in 15 L of osmotically sterilized water until a homogenous suspension was obtained and heated at 95°C for 120 min. The cell debris and precipitated E. coli proteins were discarded (2000g, 40 min, +4°C). The clear pale yellow supernatant with an OD between 0 and 1 was stored at -20°C. After thawing, an additional high-speed centrifugation was performed to remove residual cell debris and precipitated proteins in suspension: 13000g, +4°C, 20 min.

[0280] The clear, pale yellow supernatant was diluted with 10 g.L of commercial activated charcoal (Sigma-Aldrich, ref. 161551). -1The solution was stirred at 200 rpm and +22°C for 30 min at a concentration of 0.000 g. The activated carbon was then discarded by high-speed centrifugation (13000 g, 20 min, +4°C) and the supernatant was filtered through a 0.2 μm filter using a vacuum-driven filtration system (Stericup Quick Release, Millipore). For practical reasons, the clear beige supernatant was stored overnight at +4°C. The pH of the solution was then lowered to pH 4.6 using a commercial lactic acid solution (Sigma-Aldrich, ref. 27714). A white precipitate gradually appeared. The resulting suspension was centrifuged (2000 g, 20 min, room temperature) and the colorless, cloudy supernatant was discarded. The acid-precipitated casein was then resuspended in 7.5 L of osmotically sterilized water and the resulting homogeneous suspension was centrifuged (2000 g, 20 min, room temperature) and the wash water was discarded. Additional washing steps were carried out under the same experimental conditions.

[0281] The acid precipitated casein was resuspended in 750 mL of osmolyzed sterilized water. The resulting homogeneous suspension was stirred at +30° C. at 600 rpm and the pH was gradually increased to pH=7.0 by adding a homogeneous calcium hydroxide suspension at a concentration of 0.2 M. The resulting suspension was then frozen at −80° C. for 3 hours and then freeze-dried (0.002 mbar, +4° C., 60 hours). The freeze-dried calcium caseinate was stored at −20° C. for further use or analysis.

[0282] Samples at various steps of the process were analyzed by SDS-PAGE, as shown in FIG.

[0283] Example 14: Production of soft cheese replicas from recombinant caseins alpha-S1 and beta A composition of casein was prepared by co-expressing casein alpha-S1 and casein beta in the same bacteria. For this, the open reading frames P02662 and P02666 of Table 1 were cloned into the same expression vector, transformed into E. coli and produced in a fermenter using the protocol described in Example 13 (using 0.45 mm filtration instead of 0.2 mm). The casein concentration was estimated by SDS-PAGE. The preparation was estimated to have a purity of at least 95% and to contain about half of alpha-S1 casein and half of beta-casein. In all the following calculations, this composition is considered pure (therefore, the casein content should be exactly 95%).

[0284] 13 g of this dry casein composition was mixed with 39 g of water and incubated for 3 hours at 14° C. A mixture of water (15 g), cashew nuts (20 g), sunflower oil (30 g), calcium carbonate (1 g) and glucose (10 g) was blended and 22 g of this mixture was added to 45 g of the above hydrated casein composition along with 16 g of water to obtain the LpCC listed in Table 11.

[0285] [Table 11]

[0286] 80 g of this composition were placed in a mould seeded with a ferment containing lactic acid bacteria (MBT, SOGEBUL, Dole, France), yeast (DH2d SOGEBUL, Dole, France) and an additional mature ferment (PC12H, SOGEBUL, Dole, France) and incubated at a temperature of 50° C. for 16 hours.

[0287] The product was then removed from the mould and placed on a draining grid. The product and grid were placed in a box to control humidity. Table salt (La Baleine, France) was spread on the top side and the product was incubated at 14°C. After 24 hours, the product was inverted and the same salt composition was spread on the other side. The product was incubated on the grid in a purification box at 14°C to control humidity, inverted every 2 days for 6 days and then kept at 4°C. The weight of the product was estimated to be 60g with the composition given in Table 12. This composition also takes into account the degradation products resulting from lipolysis, proteolysis and sugar metabolism resulting from the activity of starter bacteria and ripening fermentants for each category. The moisture was estimated to be 68.5% on a fat-free basis. The product at day 6 is shown in Figure 15.

[0288] [Table 12]

Claims

1. 1. A method for concentrating casein in a composition containing casein and other proteins, comprising the steps of: i) providing a composition containing casein and other proteins, said composition having a pH of at least 6.5 and less than 9; ii) heating the composition so as to reduce the amount of the other proteins in a soluble fraction of the composition, wherein heating is carried out at a temperature between 75°C and 105°C; iii) recovering the soluble fraction; thereby obtaining a soluble fraction enriched in casein.

2. 1. A method for producing a casein composition comprising the steps of: i) providing a microbial composition, said microbial composition comprising a microorganism transformed with at least one nucleic acid encoding casein, said microorganism being cultured to express and produce casein, said composition having a pH of 6.5 or more and less than 9; ii) heating the microbial composition to reduce the amount of other proteins in the soluble fraction of the composition, wherein heating is carried out at a temperature between 75°C and 105°C; iii) isolating the soluble fraction from the lysed cell composition; thereby obtaining a casein composition.

3. The method of claim 1, wherein the composition comprises a microorganism transformed with at least one nucleic acid encoding casein, the microorganism is cultured to express and produce casein, and the microorganism expresses other proteins, resulting in a soluble fraction enriched in casein.

4. A method according to any one of claims 1 to 3, wherein the heating in ii) is carried out for 5 minutes to 2 hours.

5. 5. The method of claim 4, wherein the heating in ii) is carried out for at least 1 hour.

6. 4. The method according to claim 1, wherein the heating in ii) increases the proportion of casein in the soluble fraction.

7. 4. The method of claim 2 or 3, wherein the microorganisms in the microbial composition are not lysed and the heating in ii) lyses the microorganisms.

8. The method of claim 7, wherein the heating in ii) is carried out at a temperature of 80 to 100°C or 85 to 100°C.

9. 8. The method of claim 7, wherein the heating in ii) is carried out at a temperature of 95°C.

10. The method according to any one of claims 1 to 3, wherein the heating in ii) is carried out for 2 hours.

11. 4. The method according to claim 2 or 3, wherein the microorganism is transformed with one or more nucleic acids encoding two or more caseins.

12. 4. The method according to any one of claims 1 to 3, wherein the casein is selected from beta-casein, alpha-S1 casein, alpha-S2 casein and mixtures of these caseins.

13. 4. The method according to any one of claims 1 to 3, wherein the casein is selected from beta-casein, alpha-S1 casein, and mixtures of these caseins.

14. 4. The method according to any one of claims 1 to 3, further comprising the step of carrying out a further purification of the casein of the soluble fraction obtained in iii).

15. 15. The method of claim 14, wherein the further steps applied to the soluble fraction comprise at least one step selected from the group consisting of the addition of activated charcoal, membrane filtration, chromatography and casein precipitation.

16. 16. The method of claim 15, wherein the further step is acid precipitation of casein.

17. 17. The method of claim 16, wherein the further step of acidic precipitation of casein is carried out at pH=4.

18. The method of claim 2 or 3, wherein the microorganism is a bacterium.

19. The method of claim 18, wherein the bacteria is selected from E. coli, Bacillus subtilis, Salmonella typhimurium, and Lactococcus lactis.

20. A casein composition obtainable by the method according to any one of claims 1 to 3.

21. A method for obtaining curdled milk, comprising the steps of: i) providing a casein composition according to claim 20; ii) mixing said casein composition with at least one other ingredient comprising at least one component selected from the group consisting of water, calcium, lipids and carbohydrates to obtain a Liquid Pre-Curved Composition (LpCC), wherein the concentration of casein in the LpCC and the concentrations of the other ingredients in the LpCC are higher than those in milk; and iii) adding at least one coagulant to said liquid pre-curd composition to obtain curd.

22. The method of claim 21, wherein the coagulant is an acidifying agent.

23. The method of claim 22, wherein the coagulant is selected from lactic acid bacteria, lactic acid, citric acid and acetic acid.

24. 1. A method for producing an edible composition, comprising: i) carrying out the method according to claim 21; ii) further processing the curd to obtain an edible composition; A method comprising:

25. The method of claim 24, wherein the LpCC has a protein content of less than 12% (by weight) and a moisture content of more than 80% on a fat-free basis, and the edible composition has a protein content of 2-15% (by weight) and a moisture content of more than 80% on a fat-free basis.

26. The method of claim 25, wherein the LpCC has a protein content of 5-10% (by weight) and a moisture content of more than 80% on a fat-free basis, and the edible composition has a protein content of 5-15% (by weight) and a moisture content of more than 80% on a fat-free basis.

27. The method of claim 24, wherein the LpCC has a protein content of 10-18% (by weight) and a moisture content of more than 62% on a fat-free basis, and the edible composition has a protein content of 15-25% (by weight) and a moisture content of 62-80% on a fat-free basis.

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