Method for producing casein and its use

Abstract

The present invention relates to the food industry and to a novel method for producing a casein composition while limiting environmental impact by culturing transgenic microorganisms in the presence of whey, and to the use thereof, in particular for producing cheese substitutes.

Description

[Technical Field]

[0001] The present invention relates to the food industry and to a novel method for producing casein compositions and their use, particularly for producing dairy substitutes (especially vegan cheese), while limiting environmental impact. [Background technology]

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

[0003] However, dairy products are associated with several issues or concerns from a health perspective, as well as from an environmental and ethical standpoint. These concerns highlight the need for dairy alternatives that mitigate these various problems. Health issues include lactose intolerance, allergies, and high levels of saturated fatty acids, which are known to have potential adverse health effects.

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

[0005] Cattle farming is now considered one of the primary sources of GHG emissions, accounting for an estimated 7.1 gigatons of CO2 equivalent per year, or 14.5% of all man-made greenhouse gas (GHG) emissions.

[0006] Extremely high water consumption is also associated with livestock farming. Furthermore, agricultural drainage has a significant environmental impact. While the impact of crop fertilizers on groundwater should not be ignored, the impact of livestock drainage is often massive and has proven to be devastating in many parts of the world. Moreover, while livestock occupy nearly 80% of the world's agricultural land, they produce less than 20% of the world's calorie supply, indicating a very high level of stress that livestock farming places on land resources.

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

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

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

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

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

[0012] [Table 1]

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

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

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

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

[0017] For example, Escherichia coli is known to consume glucose very efficiently, but it can also consume a range of other sugars, including glucose, maltose, lactose, trehalose, fructose, xylose, and arabinose, with a set of priorities.

[0018] Many Escherichia coli strains, including BL21, are unable to consume sucrose. However, other strains such as Escherichia coli W (ATCC 9637) can efficiently use this as a carbon source (Sabri et al. (2013). Appl Environ Microbiol. 79:478-87). Sucrose consumption in Escherichia coli can be due to two gene clusters. The scr regulon encodes a sucrose phosphotransferase system (Schmid et al. (1988). Mol. Microbiol. 2:1-8), while the csc regulon encodes a phosphotransferase-independent sucrose utilization system (Jahreis et al. (2002). J. Bacteriol. 184:5307-5316). For example, Escherichia coli strains that do not consume sucrose, such as BL21, can be transformed into sucrose-consuming strains by introducing genes from, for example, the csc regulon (Bruschi et al. 2012. Biotechnol Adv. 20 ;30(5):1001-10).

[0019] The growth and metabolism of microorganisms require not only a carbon source but also a nitrogen input. For this purpose, various compounds can be added, including liquid or gaseous ammonia, urea, amino acids, peptones, and yeast extract.

[0020] Dairy-derived alternatives such as whey have also been used in fermentation with various microorganisms, including Escherichia coli (Gonzalez-Siso (1996) The biotechnological utilization of cheese whey: A review. Bioresource technology vol. 57, no 1, p. 1-11; Viitanen et al. (2003), Cheese whey-induced high-cell-density production of recombinant proteins in Escherichia coli. Microbial Cell Factories volume 2:2; Guimaraes et al. (2010) Fermentation of lactose to bio-ethanol by yeasts as part of integrated solutions for the valorisation of cheese whey. Biotechnology Advances 28: 375-384; Christensen et al. (2011) Production of bioethanol from organic whey using Kluyveromyces marxianus. J Ind Microbiol Biotechnol volume 38, pages 283-289; Pasotti et al. (2017) Cheese whey-induced high-cell-density production of recombinant proteins in Escherichia coli BMC Biotechnol. 17:48; Amaro et al. (2019) Prospects for the Use of Whey for Polyhydroxyalkanoate (PHA) Production. Front. Microbiol. Volume 10: 992-; Hausjell et al.(2019) Valorisation of cheese whey as substrate and inducer for recombinant protein production in E. coli HMS174(DE3). Bioresource Technology Reports volume 8:100340; Louaste and Elourtassi (2020) Succinic acid production from whey and lactose by Actinobacillus succinogenes 130Z in batch fermentation. Biotechnology Reports volume 27:e00481; Carranza-Saavedra et al. (2021), Kinetic analysis and modeling of L-valine production in fermentation batch from E. coli using glucose, lactose and whey as carbon sources. Biotechnology Reports volume 31, e00642; Chaparro et al. (2021), Whey as an Alternative Nutrient Medium for Growth of Sporosarcina pasteurii and Its Effect on CaCO3 Polymorphismand Fly Ash Bioconsolidation. Materials volume 14: 2470; Mobayed et al. (2021) Effect of by-products from the dairy industry as alternative inducers of recombinant β-galactosidase expression. Biotechnol.Letter 43(3):589-599; Zou and Chang (2022) Past, Present, and Future Perspectives on Whey as a Promising Feedstock for Bioethanol Production by Yeast. Journal of Fungi, volume 8, pages 395-; de Diviitis et al. (2023) Cheese-whey permeate improves the fitness. Biotechnology for Biofuels and Bioproducts, volume 16:30). .

[0021] Whey is a by-product or waste product of the cheese-making process, but it is also present in other processes such as Greek yogurt production, where some whey is discharged from the product after coagulation. Cheese production can yield 10 liters of whey per kilogram of cheese (Mollea et al. (2013) Chapter 24: Valorisation of cheese whey, a by-product from the dairy industry. Pages 549-588 in Food Industry. I. Mazzalupo, ed. InTech Open, London, UK), while the production of 1 kg of Greek yogurt can yield approximately 2-3 kg of acidic whey (Erickson, BE (2017) Chem. Eng. News, 95(6), 26-30).

[0022] The valorization of whey, particularly acid whey, is an important issue in the dairy industry (Gonzales-Siso (1996) The biotechnological utilization of cheese whey: a review. Bioresour Technol. 57:1-11; Marwaha and Kennedy (2007) Whey-pollution problem and potential utilization. Int J Food Sci Technol.;23:323-36; Rocha-Mendoza et al (2021) Acid whey trends and health benefits. J. Dairy Sci. volume 104, pages 1262-1275). Some companies may have the potential to valorize whey (particularly, in some cases, not only sweet whey but also acid whey), but in many cases, acid whey is discarded as waste.

[0023] Whey contains proteins (whey proteins, about 20% of the total milk proteins) and has a complex composition including minerals and lactose. Whey is characterized by a greenish-yellow color due to the presence of riboflavin (vitamin B12; Gonzalez-Siso (1996) The biotechnological utilization of cheese whey: A review. Bioresource technology vol. 57, no 1, p. 1-11;). Indeed, whey contains various minerals including calcium, phosphorus, magnesium, sodium, and potassium, and is also rich in water-soluble vitamins such as B1 (thiamine), B2 (riboflavin), B6 (pyridoxine), B12 (cobalamin), and C (ascorbic acid). There are mainly two types of whey: sweet whey and acid whey.

[0024] Sweet whey is a by-product associated with most cheese production after enzymatic coagulation using natural or synthetic rennet. It has a pH of approximately 6–7 (Gonzalez-Siso (1996) The biotechnological utilization of cheese whey: A review. Bioresource technology vol. 57, no 1, p. 1–11), a higher protein content, and lower salt content than acidic whey.

[0025] Acid whey is associated with the acid coagulation of milk. Acid coagulation can result from the activity of lactic acid bacteria, which lower the pH by producing organic acids, or from the addition of organic acids (lactic acid, citric acid, acetic acid) or inorganic acids (sulfuric acid, hydrochloric acid). Acid whey is a natural byproduct of cottage cheese and Greek yogurt, two products whose production has increased dramatically. While its value-added production is challenging for several inherent reasons (Rocha-Mendoza et al (2021) Acid whey trends and health benefits. J. Dairy Sci. volume 104, pages 1262-1275), its use in fermentation offers a solution to address this challenge.

[0026] The composition of whey can vary depending on the manufacturing method. For example, in acidic whey, the lactose concentration can vary in the range of approximately 2-5% (weight / volume) (Rocha-Mendoza et al (2021) Acid whey trends and health benefits J. Dairy Sci. volume 104, pages 1262-1275; O'Donoghue and Murphy (2023) Comprehensive Reviews in Food Science and Food Safety, 22:2652-2677).

[0027] Various whey derivatives can be obtained to add value to whey protein and lactose.

[0028] The whey permeate is obtained after removing whey proteins using ultrafiltration (O'Donoghue and Murphy (2023), see above). The whey permeate can be used as a source of milk solids in various food, beverage, or feed applications.

[0029] In the delactated whey permeate, most of the lactose has been removed, but lactose can still account for 60% of the dry weight and therefore continues to make up a significant portion of the remaining composition.

[0030] Whey can also be concentrated to facilitate transport.

[0031] The inventors refer to such products as whey or whey, regardless of whether they are untreated (or whole) whey, permeate, or delactated permeate, as long as they still contain at least about 2% (weight / weight, wet weight), preferably at least 3%, preferably at least 4%, preferably at least 5%, and preferably at least 8% lactose. In some embodiments, when concentrated whey is used, it may contain at least 30% (weight / weight, wet weight) lactose, or at least 32% lactose, or 35% or more lactose.

[0032] Using this type of raw material is unsuitable for producing animal-free products, given the origin of the whey. Nevertheless, using waste as a raw material can be very useful in achieving good economics and a favorable life cycle analysis.

[0033] During feed-batch fermentation, the pH can be adjusted to maintain it within a specific range, and for this purpose, acidic or basic compounds including hydrochloric acid, sulfuric acid, lactic acid, and phosphoric acid (acidic compounds) or liquid or gaseous ammonia, sodium hydroxide, or potassium hydroxide (basic compounds) can be added.

[0034] All of these solutions are not mutually exclusive and can be combined.

[0035] The inventors proposed using whey as a carbon source for culturing casein-expressing microorganisms, particularly in fed-batch culture. They demonstrated that using such a product reduces environmental impact compared to the effects observed when glucose, sucrose, or sucrose molasses are used as raw materials for microbial growth (with the use of isopropyl β-D-1-thiogalactopyranoside (IPTG), where appropriate, to induce casein production). As described above, given the composition of whey, using such a product also provides vitamins and minerals to the culture medium, which is of interest for maintaining long-term cell (especially bacterial) cultures.

[0036] The fed-batch process is a method of culturing microorganisms such as bacteria, yeast, or cells in a bioreactor to produce a desired substance. This process allows for greater control of the culture environment and can result in higher yields and product concentrations compared to batch culture.

[0037] The process begins with inoculating a small amount of microorganisms (inoculum) into a bioreactor containing a growth medium. This medium contains essential nutrients (such as sugars, nitrogen sources, minerals, and vitamins) necessary for the microorganisms to grow and produce the desired product.

[0038] The bioreactor initially operates in batch mode, adding all necessary nutrients at the start of the culture. During this phase, microorganisms grow, consume nutrients, and may also produce the desired product. The culture medium should not contain excess nutrients to avoid overgrowth, the production of harmful by-products by the microorganisms, and to ensure better control.

[0039] As cultivation progresses, the concentration of nutrients in the bioreactor decreases due to consumption by microorganisms. During the fed-batch phase, additional nutrients are gradually introduced into the bioreactor. To maintain optimal growth conditions and extend the production phase, the rate and amount of nutrient addition, as well as various parameters such as temperature, pH, dissolved oxygen, stirring speed, and nutrient supply rate, are strictly monitored, controlled, and adjusted as needed to optimize cell growth and product formation based on factors such as cell density, nutrient levels, and product formation. In a continuous fed-batch process, nutrients are supplied continuously. In other fed-batch processes, nutrients are supplied as needed.

[0040] The culture is harvested when the desired product concentration is achieved or after a predetermined period. Harvesting is done before the amount of microorganisms decreases, as the death of microorganisms can lead to the formation of undesirable products. The product can be purified and further processed.

[0041] The advantages of the fed-batch process include control over microbial growth and product formation by adjusting the rate of nutrient delivery, higher yields through controlled nutrient delivery, extended production stages, and reduced by-product generation.

[0042] In one embodiment, whey is present in the culture medium at the start of the culture (inoculation stage) either alone or preferably with another sugar, particularly glucose, sucrose, or fructose, especially in the form of molasses.

[0043] In another embodiment, whey is not present in the culture medium at the start of the culture (inoculation stage). In this embodiment, other sugars, particularly glucose, sucrose, and fructose, are present in the culture medium, especially in the form of molasses.

[0044] In one embodiment, whey is introduced as a carbon source for at least part of the feeding period. In one embodiment, no other sugars are provided with the whey. In another embodiment, whey is introduced with other sugars as described above.

[0045] In one embodiment, whey is present in a starter culture medium along with sugars other than lactose (particularly glucose), and the feed-add process includes providing additional nutrients, and for at least a certain period (15 minutes to 1 hour, preferably 20 minutes to 45 minutes, preferably about 30 minutes), no sugars are provided along with the additional nutrients, after this period, sugars other than lactose (particularly glucose) are provided again along with the additional nutrients. In one embodiment, whey is not further provided along with sugars other than lactose (particularly glucose). In another embodiment, whey is further provided along with sugars other than lactose (particularly glucose).

[0046] In one embodiment, whey is not present in the starter culture medium containing only sugars other than lactose (particularly glucose). In this embodiment, the feed-add process includes the provision of additional nutrients, and whey is the only sugar provided with the additional nutrients for at least a certain period (15 minutes to 1 hour, preferably 20 minutes to 45 minutes, preferably about 30 minutes). In this embodiment, after this period, sugars other than lactose (particularly glucose) may be provided again along with additional nutrients. In one embodiment, whey is not further provided with sugars other than lactose (particularly glucose). In another embodiment, whey is further provided with sugars other than lactose (particularly glucose).

[0047] One objective is to utilize lactose, present in whey, as the sole sugar used by microorganisms for a certain period during cell culture. If casein expression is induced by lactose, this also ensures a switch to casein production by the microorganisms. Once casein production has begun, it is possible to reintroduce glucose or another sugar (even lactose as a sugar).

[0048] In view of the lactose concentration in unconcentrated whey, one preferred embodiment is to use a starter medium containing whey (60% or less, preferably 55% or less, particularly about 45% to 55%, particularly about 50% (vol / vol)) and also containing other sugars that induce CCR (see below), such as glucose. The microorganisms then use and consume the other sugars. At a time specified by those skilled in the art (usually when the microorganisms are in the later stages of logarithmic growth), no further sugars other than lactose are provided, and as a result, the microorganisms consume the lactose that has been present in the medium since the start of the culture (which was not consumed because the other sugars were used as a carbon source by the microorganisms). Thus, lactose catabolism is initiated and CCR is released (thus enabling the consumption of lactose present in the medium and, if lactose-inducible, the induction of casein production). Once the lactose is consumed, further carbon supply can be obtained by supplying any of the sugars (glucose, lactose, fructose, sucrose) without further provision of whey.

[0049] In some embodiments, whey is used as the sole sugar source (lactose present in the whey).

[0050] In other embodiments, whey is used with other ingredients such as glucose (which can be obtained from corn or wheat) and sucrose (which can be obtained from sugar beets or sugarcane). Such other ingredients may be pure sugar, molasses, or other forms.

[0051] It is preferable to use whey together with other ingredients. In fact, because the lactose concentration in whey is generally low, if such a product is used as the sole ingredient, a large amount of whey must be added to obtain a sufficient amount of lactose.

[0052] In addition, since whey contains lactose, when a lactose-inducible promoter is used, it can induce the expression of a transgene that expresses casein (Viitanen et al. (2003), Cheese whey-induced high-cell-density production of recombinant proteins in Escherichia coli. Microbial Cell Factories volume 2:2; Hausjell et al. (2019) Valorisation of cheese whey as substrate and inducer for recombinant protein production in E. coli HMS174(DE3). Bioresource Technology Reports volume 8:100340; Mobayed et al. (2021) Effect of by-products from the dairy industry as alternative inducers of recombinant β-galactosidase expression. Biotechnol. Letter 43(3):589-599; de Diviitis et al. (2023) Cheese-whey permeate improves the fitness. Biotechnology for Biofuels and Bioproducts, volume 16:30;

[0053] In one embodiment, it is proposed that the culture medium contain both glucose and whey during the initial stages of microbial growth (until the end of logarithmic growth or the start of steady-state growth), while glucose is preferred for carbon catabolism inhibition (CCR), and once adequate microbial growth is achieved and recombinant casein expression is desired, the focus shifts to lactose catabolism (Gorke and Stulke Nat Rev Microbiol 6, 613-624 (2008); Postma et al Microbiol Rev. 1993 Sep;57(3):543-94). It is possible to supply low levels of glucose to the culture, which maintains adequate carbon supply to maintain the vitality of microbial growth without cleavage to glucose-only use, and thus allows protein expression to continue. While other sugars (such as sucrose or galactose) can also be used, the use of glucose is preferred, at least at the start of culture (logarithmic growth), to induce CCR, particularly when a lactose-inducible promoter is used to induce casein expression.

[0054] Lactose is a disaccharide composed of glucose and galactose. Its metabolism typically relies on hydrolysis to glucose and galactose. Some Escherichia coli strains, such as BL231(DE3), are unable to consume the galactose portion due to mutations that result in a lack of enzymes involved in galactose metabolism via the Leloir pathway. Consequently, to fully utilize lactose as a carbon donor to bacteria with maximum yield, it is possible and preferable to use strains proficient in the Leloir pathway (Hausjell et al. (2019) Valorisation of cheese whey as substrate and inducer for recombinant protein production in E. coli HMS174(DE3). Bioresource Technology Reports volume 8:100340) or to repair this pathway in mutant strains through genetic engineering. Such strains capable of metabolizing D-galactose to form glucose are generally designed as gal+ strains.

[0055] Furthermore, the inventors used a refined purification procedure that not only produces casein with a high level of purity and a grade suitable for use in the food industry, but is also easily scalable to an industrial level and has a cost, particularly energy cost, that is suitable for development.

[0056] In particular, recombinant casein can be isolated from biomass and culture media by heating the composition, as disclosed in International Publication No. 2022 / 253816.

[0057] Using the methods disclosed herein makes it possible to obtain casein compositions with reduced environmental impact, particularly compared to glucose, compared to the effects observed when glucose, sucrose, or sucrose molasses are used as a carbon source for culturing microorganisms (i.e., when whey, especially acidic whey or acidic whey permeate, is not used in the methods). In addition, this makes it possible to provide the dairy industry with a method for reusing waste that currently has to be treated and disposed of. When microorganisms are used with lactose-induced casein expression of casein, IPTG is used to induce protein expression when casein expression is induced by lactose in whey.

[0058] Environmental impacts are multifactorial and must be considered from the perspectives of climate change, freshwater, seawater, soil and air pollution, impacts on ecosystems, fossil fuel use, depletion of material resources, water use, and other aspects. Criteria include: (i) Measured by climate change (kgCO2eq, eq stands for equivalent) (ii) Acidification, molar H + -eq (iii) Ecotoxicity, comparative toxicity units (CTUe) for ecosystems (iv) Use of non-renewable energy resources (MJ or kWh) (v) Eutrophication of freshwater (kg P-eq) (kg phosphorus equivalent) (vi) Eutrophication of seawater (kg N-eq) (kg nitrogen equivalent) (vii) Eutrophication of terrestrial areas (cumulative excess, molar N-eq) (viii) Toxicity and carcinogenicity to humans (comparative toxicity units to humans, CTUh) (ix) Toxicity to humans, non-carcinogenicity (comparative toxicity units to humans, CTUh) (x) Ionizing radiation, effects on human health (U 235 Relative human exposure efficiency relative to kNq U 235 -eq) (xi) Land use, soil quality index (ss) (xii) Use of material resources (kg Sb-Eq) (xiii) Ozone depletion potential (kg CFC11-eq) (xiv) Formation of particulate matter (disease incidence) (xv) Formation of photochemical oxidants (kg NMVOC-eq) (xvi) Water usage (m 3 World EQ Deprived One could list these:

[0059] This method is interesting because it can provide improvements to at least one of these criteria, particularly climate change, acidification, eutrophication (v, vi and / or vii), and / or water use. In particular, it can achieve improvements to climate change impacts and at least one other criterion.

[0060] Therefore, the present invention relates to a method for reducing the environmental impact of a casein composition prepared by fermentation of transgenic microorganisms, a. Prepare microorganisms transformed with at least one nucleic acid encoding casein, b. Culturing the microorganism using a medium containing whey to express and produce casein, c. This provides a microbial composition in which the pH of the composition is 6.5 or higher, preferably less than 9. d. Heating the microbial composition to reduce the amount of other proteins in the soluble fraction of the composition, wherein the heating is carried out at a temperature of 75°C or higher. e.iv) The soluble fraction is recovered from the heated cell composition, thereby obtaining a casein composition in the soluble fraction, and the environmental impact is reduced compared to the environmental impact of the casein composition obtained by the same process, except that glucose, sucrose, or molasses is used instead of whey in b. This also includes methods.

[0061] In particular, the reduction of environmental impacts is observed in at least one criterion selected from (i) climate change, (ii) acidification, (iii) ecotoxicity, (iv) use of non-renewable energy resources, (v) eutrophication of freshwater, (vi) eutrophication of seawater, (vii) eutrophication of terrestrial areas, (viii) toxicity to humans, carcinogenicity, (ix) toxicity to humans, non-carcinogenicity, (x) ionizing radiation, effects on human health, (xi) land use, (xii) use of material resources, (xiii) ozone depletion potential, (xiv) formation of particulate matter, (xv) formation of photochemical oxidants, and (xvi) water use, preferably (i) climate change.

[0062] Casein compositions can be processed to obtain not only dairy substitutes such as cheese and yogurt resulting from coagulation, but also ice cream, reconstituted milk, and similar products. The environmental impact of such dairy substitutes is reduced compared to the environmental impact observed when recombinant casein derived from microorganisms grown in the presence of glucose, sucrose, or sucrose molasses as a carbon source is used. This generally results in a more favorable environmental impact in all respects compared to the same dairy substitute made using recombinant casein derived from microorganisms grown in the presence of glucose alone as the carbohydrate source. Even when recombinant casein derived from microorganisms grown in the presence of sucrose, or even in the presence of sucrose molasses, the environmental impact is improved in many respects.

[0063] In this invention, "casein" refers to any casein protein or a mixture of casein proteins. Therefore, "casein" is alpha-S1 casein, alpha-S2 casein, beta-casein, or kappa-casein. To some extent, this may also refer to a mixture of any of such proteins. The terms "casein" or "caseins" can be used to refer to casein proteins in general.

[0064] In this invention, the term "between" includes the limit value.

[0065] In this invention, the term "dairy substitute" means a food product that has the essential characteristics of dairy products (such as cheese, yogurt, ice cream, etc.) obtained using cow's milk in terms of nutritional value, appearance, texture, and taste.

[0066] In this invention, the term "cheese substitute" means a food product that has the essential characteristics of cheese in terms of nutritional value, appearance, texture, and taste.

[0067] The term "fresh cheese" refers to cheese with a fat-free base that has a moisture content of more than 80% (the percentage of water relative to the total mass of the fat-free product) and a protein content of 2% to 15% of the total weight (the percentage of protein relative to the total mass of the product).

[0068] The terms "soft cheese" or "semi-soft cheese" refer to cheeses with a fat-free base moisture content of 62% to 80% (the percentage of water relative to the total mass of the product) and a protein content of 15% to 30% of the total weight (the percentage of protein relative to the total mass of the product). Soft cheeses have a fat-free base moisture content of 67% to 80%, while semi-soft cheeses have a fat-free base moisture content of 62% to 67%.

[0069] In the present invention, the term “liquid pre-curd composition” or “LpCC” refers to a composition containing at least casein and at least one other component, including at least one of water, calcium, lipids, and carbohydrates, prior to the addition of rennet and fermentation and coagulation (particularly casein coagulation). The casein concentration in LpCC is higher than that in milk. The concentrations of the other components in LpCC are also higher than those in milk. This allows for some water savings when using LpCC containing recombinant casein as disclosed herein, since LpCC does not represent artificial (cow's) milk. The concentrations of proteins, lipids, calcium salts, and / or carbohydrates are adjusted to suit the final desired composition of the cheese substitute.

[0070] In this invention, the term "coagulant" refers to a chemical or biochemical composition capable of causing coagulation. Coagulation can be achieved by adding an acid solution, adding a starter culture (a fermented product whose growth in the presence of carbohydrates causes a decrease in pH), heat treatment, adding a calcium chelating agent, adding natural or recombinant rennet, adding a rennet substitute (such as an animal protease or a plant-derived coagulating enzyme), or a combination of these steps. The coagulant may be any additive, compound, composition, or treatment, used alone or in combination with another or other additive, compound, composition, or treatment, whose addition results in coagulation. When preparing animal-free food compositions, it is preferable to use an acidifying agent (such as an acid or starter culture) so that the coagulant does not contain any animal-derived elements, in order to avoid the use of rennet. Acidic chemicals can also be used, although it is advantageous if the acidifying agent contains lactic acid bacteria.

[0071] In the present invention, the term "curd" means a composition in which casein coagulates or precipitates under the action of a coagulant, which, if present, can be separated from the liquid phase, for example, by draining on a cheesecloth. The curd also includes other components, including water and lipids (if present).

[0072] In this invention, the term "fermented product" refers to a composition containing at least one microbial strain that is added during the production of a cheese substitute. The lactic acid fermented product is responsible for lactic acid fermentation, particularly resulting in the acidification of the culture medium. As a result, these can be used as coagulants.

[0073] Other fermented products are used in cheesemaking for curd processing, resulting in changes to texture, taste, aroma, and chemical composition (particularly involving the cleavage of proteins into smaller peptides). These fermented products can be called “aging fermented products,” “matured fermented products,” or “mature fermented products” and are not typically used in the production of fresh cheese. Such aging fermented products can be added along with coagulants.

[0074] In this invention, the term "non-animal" means a compound or composition that is not obtained directly from an animal, is not produced from animal cells in culture, and is not isolated from animal products such as milk. Therefore, a compound or composition produced by microbial fermentation is "non-animal," even if some animal-derived products, such as bactopeptone, may be involved in the fermentation. Accordingly, in this invention, a protein naturally produced in an animal is called non-animal if it is produced within a microbial (such as a bacterium or yeast) cell or plant cell, even if its sequence or structure may be identical to that of a protein that would be isolated from an animal.

[0075] In the present invention, "animal-free" means a compound or composition that is not obtained from animals, from animal cells in culture, or from animal products such as milk, and whose manufacturing method does not involve any animal-derived raw materials or additives.

[0076] In the present invention, the term "texturing agent" means any gelling agent, including emulsifiers such as lecithin, and hydrocolloids such as cassia gum, sesbania gum, tamarind gum, guar gum, fenugreek gum, gum arabic, agar (or agar-agar), carrageenan, tragacanth gum, xanthan gum, carob (locust bean) gum, and cellulose gum.

[0077] In a first aspect, a method for obtaining a casein composition is described herein. i. Prepare microorganisms transformed with at least one nucleic acid encoding casein, ii. Culturing the microorganism using a medium containing whey to express and produce casein, iii. This provides a microbial composition having a pH of 6.5 or higher, preferably less than 9, iv. Heating the microbial composition to reduce the amount of other proteins in the soluble fraction of the composition, wherein the heating is performed at a temperature of 75°C or higher. The soluble fraction is recovered from the heated cell composition of v.iv), thereby obtaining the casein composition in the soluble fraction. The present invention discloses methods including the following:

[0078] During step (ii), the whey may be used in combination with other sugars such as glucose, saccharose, or refined lactose.

[0079] The method described above allows for a reduction in environmental impact for at least one of the criteria listed above (in particular, greenhouse gas (GHG) emissions by mass of CO2eq) compared to when the method is carried out using glucose, sucrose, or sucrose molasses instead of whey in step (ii). The impact can be reduced in particular when acidic whey (or a derivative such as permeate) is used as whey in step (ii).

[0080] In particular, compared to glucose use, it is possible to achieve reductions of at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, or in some cases at least 30%, depending on the criterion of focus.

[0081] A reduction in environmental impact can also be observed in dairy substitutes made from casein obtained by the methods disclosed herein.

[0082] It should be noted that microorganisms, particularly bacterial cells, exhibit a logarithmic (or log) phase in which the number of cells doubles after each generation. During this phase, metabolic activity is high, and the energy (nutrients) in the culture medium is directed towards this doubling activity. After the logarithmic phase, the population growth experienced during the log phase begins to decline as cell growth plateaus or reaches a stationary phase, and the number of dividing cells equals the number of dead cells.

[0083] In connection with the methods disclosed herein, it is preferable to have whey as a raw material used by the microorganism, preferably in the mid-to-late logarithmic growth phase or at the start of the stationary phase (when avoiding the supply of sugars other than lactose and supplying lactose, or when whey is present in the culture medium from the inoculation stage), and optionally to maintain this supply during casein production. However, an initial induction step may also be envisioned. Those skilled in the art can determine the end of the logarithmic phase or the start of the stationary phase for microbial culture, depending on the doubling time of the microorganism, the concentration of the starter culture, and the duration of growth. Measuring the optical density (OD) of the culture may also provide information about the growth stage of the microorganism. When using Escherichia coli as the microorganism, a logarithmic phase of 16-20 hours, followed by a stationary phase of about 7-10 hours in which protein expression occurs, can be envisioned.

[0084] The microbial composition is a liquid solution containing casein and other proteins. It also presents soluble and insoluble fractions. This composition also contains transgenic microorganisms, such as bacteria that produce casein as a result of the presence of a transgene encoding casein in their genome. Such bacteria are present in the insoluble fraction (whether lysed or not).

[0085] Compositions containing transgenic microorganisms are obtained by fermentation using various sugars and nitrogen sources as raw materials. In one embodiment, fermentation is carried out in a medium containing glucose, sucrose, or lactose in addition to whey. In a preferred embodiment, glucose, sucrose, or lactose is used as a carbon source. In some embodiments, fermentation is carried out in a medium using molasses or vinas as a carbon source. In one embodiment, fermentation is carried out in a medium containing molasses as a carbon source. In another embodiment, liquid or gaseous ammonia, urea, amino acids, peptone, or yeast extract is present in or added to the medium as a nitrogen source. In one embodiment, fermentation is carried out in a medium containing ammonia, urea, amino acids, peptone, or yeast extract as a nitrogen source. In one embodiment, fermentation is carried out on a medium containing whey as the main raw material (main carbon source). In one embodiment, fermentation is carried out in a medium containing acidic whey or acidic whey permeate. In one embodiment, fermentation is carried out on a medium containing whey and another sugar as an additional sugar source. In preferred embodiments, fermentation is carried out on a culture medium containing acidic whey (or acidic whey permeate) as an additional sugar source, along with glucose, sucrose, or lactose. Glucose can be used as another carbon source.

[0086] The pH can be adjusted by adding liquid or gaseous ammonia, sodium hydroxide, potassium hydroxide, calcium hydroxide, or other basic compounds before heating or during microbial growth. The pH should be greater than 6.5, preferably less than 9.

[0087] Microorganisms are transgenic and contain at least one transgene (nucleic acid) that encodes casein introduced into the microorganism. This transgene may be located within the microbial genome or outside the genome (on an artificial chromosome such as a plasmid, cosmid, bacterial artificial chromosome, or yeast artificial chromosome).

[0088] In certain embodiments, the microorganism is a bacterial cell, particularly Escherichia coli. In other embodiments, the microorganism is a fungal cell (including yeast cells). In yet another embodiment, the microorganism is a eukaryotic cell, particularly a plant cell. In particular, suitable prokaryotic hosts for expressing casein include Escherichia coli, Bacillus subtilis, Salmonella typhimurium, Lactococcus lactis, and various species within the genera Lactococcus, Pseudomonas, Streptomyces, and Staphylococcus. Suitable eukaryotic hosts for casein expression include fungi such as Saccharomyces cerevisae, Kluyveromyces lactis, Pichia pastoris, or Trichoderma reesei. Plant cells may also be used for protein production.

[0089] Casein is preferably produced in non-animal cells, more preferably in bacterial cells.

[0090] The microorganism is transformed with at least one nucleic acid to express casein. Such nucleic acid contains elements necessary for the transcription and translation of the transgene encoding casein. In particular, the nucleic acid shall contain a promoter sequence, a casein-coding sequence, and a terminator sequence.

[0091] In some embodiments, the microorganism is transformed with a single nucleic acid encoding a single casein (and produces such casein). Preferably, the expression of the nucleic acid (protein production) is induced by lactose, i.e., the nucleic acid encoding the casein is expressed in the presence of lactose metabolized by the microorganism (lactose-induced expression).

[0092] In other embodiments, the microorganism expresses two caseins. In such embodiments, the microorganism may be transformed with two transgenes, each expressing a predetermined casein. It is preferable that the expression of at least one transgene is lactose-induced. Preferably, the expression of both transgenes is lactose-induced (using the same or different systems). In another embodiment, the two transgenes are linked to each other in an operon (thus, caseins are produced simultaneously). It is preferable that the expression in the operon is lactose-induced.

[0093] Furthermore, embodiments in which the microorganism expresses more than two caseins are also conceivable. In such embodiments, each transgene encoding a given casein may be under the control of its own promoter (preferably for lactose-induced expression), or some or all of the transgenes may be co-expressed in an operon (preferably for lactose-induced expression).

[0094] In some embodiments, the microbial culture contains a mixture of several microorganisms transformed with one nucleic acid encoding one casein and other microorganisms transformed with one nucleic acid encoding another casein.

[0095] Lactose-induced gene expression is known in the art. This is often based on a lactose operon (lac operon), which contains an operator region (lac o) to which the lac repressor lacI binds in the absence of lactose, thereby preventing transcription of the gene downstream of the lac o region. In the presence of lactose (or allolactose, an isomer of lactose), the conformation of the lacI protein changes so that it no longer binds to lac o, thereby enabling transcription of the sequence downstream of the operator. The sequence of the lac operator is TTGTGAGCGGATAACAA (SEQ ID NO: 5). The system (including the lac o operator and lacI repressor) can be used for the expression of genes downstream of the lac o operator, provided that the promoter upstream of the lac o operator is either the native promoter of the lac operon or another promoter (such as the T7 promoter). The lac operon includes a regulatory region containing a promoter (P) to which RNA polymerase binds and initiates transcription, an operator (O) to which a lac repressor binds and prevents transcription of structural genes in the absence of lactose, and a lacI gene encoding a lac repressor that binds to the operator and blocks transcription in the absence of lactose.

[0096] It can be noted that lactose-inducible promoters and systems have been described in the art. Examples include the lac promoter, which regulates the expression of the lactose operon (lac operon), an operon necessary for lactose transport and metabolism in Escherichia coli (E. coli) and many other enterobacteria. Also, there are the P(bgaL) promoter (Hartman et al, Appl Environ Microbiol. 2011 Jan;77(2):471-8) and the P(bgaL) promoter disclosed in Zhang et al (Biochemical Engineering Journal Volume 151, 15 November 2019, 107316). lacA and P lacLMPromoter, or P disclosed in Heiis et al (Microb Cell Fact 15, 50 (2016)) lacA Promoters can also be mentioned.

[0097] In some embodiments, microorganisms can use galactose as a carbon source. Particularly preferred is when the microorganism expresses the galETKM operon derived from the E. coli K12 strain, which is necessary for galactose consumption. It should be recalled that the galETKM operon contains four genes; galE encodes epimerase, galT encodes uridilyltransferase, galK encodes galactokinase, and galM promotes the interconversion between β-galactose and α-galactose.

[0098] Therefore, in some embodiments, casein is cultured in bacteria under the control of a lactose-inducible promoter (e.g., a promoter fused to the laco region, and bacteria expressing the lacI repressor), and the bacteria also possess the genetic machinery necessary for galactose consumption. Thus, in this embodiment, the bacteria can efficiently consume glucose, lactose (after CCR is complete), and galactose produced from lactose consumption.

[0099] In the embodiment, The preparation of a microorganism transformed with at least one nucleic acid encoding casein, wherein the nucleic acid is under the control of a lactose-inducible promoter, Preferably, the microorganisms are cultured in the presence of glucose and, if necessary, whey until the logarithmic growth phase. In particular, when microorganisms are in the logarithmic growth phase, reducing glucose levels is important. To culture microorganisms using a whey-containing medium to induce the expression of the gene encoding casein and the production of casein (glucose depletion will abolish the catabolic repression of the lactose-inducible promoter), If necessary, after inducing casein expression and production, the microorganisms may be further cultured in the presence of glucose. If necessary, the composition of cultured microorganisms expressing and producing casein may be recovered. A method for culturing microorganisms containing casein can be cited. This method involves various steps described elsewhere in this specification. It allows for inducing casein expression when the cells are adapted to such expression (essentially during the logarithmic growth phase, around the mid-stage of this phase), and for providing the cells with the best carbon source while reducing costs and environmental impact. Thus, a culture of casein-expressing microorganisms (preferably bacteria) will be obtained.

[0100] especially, i. Preparing a microorganism transformed with at least one nucleic acid encoding casein, wherein the expression of the nucleic acid is induced by lactose, ii. Culturing the microorganism in a culture medium containing glucose as a carbon source, wherein nucleic acid expression is not induced. iii. When microorganisms are in logarithmic growth, glucose is depleted from the culture medium, and at the time of depletion, whey is present in the culture medium, thereby providing lactose present in the whey as a carbon source, and nucleic acid expression is induced by the lactose present in the whey. iv. Further culturing of microorganisms in the presence of a carbon source, A method for culturing microorganisms containing [the specified substance] can be performed.

[0101] In one embodiment, glucose is not present as a carbon source in step iv.

[0102] In one embodiment, whey is not present as a carbon source in step iv.

[0103] In one embodiment, the carbon source in iv includes both glucose and whey.

[0104] In one embodiment, as disclosed elsewhere, the microorganism also metabolizes galactose as a carbon source.

[0105] In one embodiment, the nucleic acid encoding casein is under the control of a promoter fused to the laco operator region of the lactose operon, and the microorganism expresses the lacI repressor of the operon. This ensures catabolic suppression of expression in ii. Therefore, in this embodiment in particular, glucose and whey can be present in the culture medium of ii.

[0106] In one embodiment, whey is not present in the culture medium of ii, but is added in iii.

[0107] In one embodiment, glucose depletion in iii occurs through the consumption of glucose by microorganisms without the replenishment of glucose (in fact, it is generally necessary to continuously supply glucose or another carbon source during cultivation, and stopping this replenishment leads to the consumption of all previously supplied glucose, causing the microorganisms to begin using whey as a carbon source).

[0108] In one embodiment, the microorganism is a bacterium, particularly Escherichia coli (E. coli).

[0109] This method makes it possible to obtain a casein composition when it includes the following steps: The present invention provides a microbial composition having a pH of 6.5 or higher, preferably less than 9. Heating the microbial composition to reduce the amount of other proteins in the soluble fraction of the composition, wherein the heating is performed at a temperature of 75°C or higher. The soluble fraction was recovered from the heated cell composition. This results in obtaining a casein composition in the soluble fraction.

[0110] As described above, certain embodiments include situations in which the initial composition (a composition containing casein and other proteins) is obtained from a bacterial culture. In such embodiments, as described above, the bacteria are transformed with one or more nucleic acids encoding one or more caseins.

[0111] In particular, in one embodiment, bacteria are transformed with one or more nucleic acids encoding beta-casein.

[0112] In particular, in one embodiment, the bacteria are transformed with one or more nucleic acids encoding alpha-S1 casein.

[0113] In particular, in one embodiment, the bacteria are transformed with one or more nucleic acids encoding alpha-S2 casein.

[0114] In particular, in one embodiment, bacteria are transformed with one or more nucleic acids encoding beta-casein and alpha-S1 casein.

[0115] In particular, in one embodiment, the bacteria are transformed with one or more nucleic acids encoding beta-casein, alpha-S1 casein, and alpha-S2 casein, or a combination of two of these.

[0116] It should be noted that when multiple proteins are produced, bacteria can be transformed with different nucleic acids (each encoding a different protein) or with a single nucleic acid (containing elements such as operons that enable the production of various proteins). Methods for such transformation of microorganisms, and especially bacteria, to produce one or more proteins are known in the art.

[0117] As a result, the recovered casein composition contains casein, preferably selected from beta-casein, alpha-S1 casein, alpha-S2 casein, and mixtures of these caseins, and particularly selected from beta-casein, alpha-S1 casein, and mixtures of these caseins. As shown, the microorganisms are transformed with nucleic acids encoding alpha-casein and another nucleic acid encoding beta-casein, and the two genes are under the control of a lactose-inducible promoter.

[0118] It is preferable that the microorganisms have not been transformed with nucleic acids encoding kappa-casein. As a result, it is preferable that kappa-casein is not produced by the microorganisms and that kappa-casein is not present in the various compositions and solutions.

[0119] In the methods disclosed herein, whey serves as an important carbon source necessary for the growth of microorganisms.

[0120] In one embodiment, as described above, the culture of microorganisms is initiated in the presence of sugars other than lactose, particularly glucose. When bacteria, especially Escherichia coli (E. coli), are used as the microorganisms, glucose is present in sufficient quantities to induce carbon catabolism repression (CCR). Indeed, the presence of both glucose and lactose results in a regulatory scenario in which the lac operon is repressed. High priority is given to glucose utilization via catabolism repression, which keeps cAMP levels low and essentially reduces or eliminates the transcriptional activity of the lac operon.

[0121] In this type of development, bacteria use glucose as a carbon source, and lactose metabolism is suppressed. Recall that lactose metabolism involves the cleavage of one molecule of lactose into one molecule of glucose and one molecule of galactose. As a carbon source in the culture medium, the catabolic products produced by the breakdown of glucose inhibit the activation of enzymes necessary for lactose metabolism, thus suppressing this metabolism in the presence of glucose. However, once these enzymes are activated, suppression no longer occurs (Gorke and Stulke Nat Rev Microbiol 6, 613-624 (2008); Postma et al. Microbiol Rev. 1993 Sep;57(3):543-94).

[0122] In particular, it is preferable to use glucose (or a sugar other than lactose) at the start of culture and during the initial logarithmic phase of growth to ensure proper logarithmic growth of bacteria.

[0123] Whey shall be used as a lactose source to induce casein expression (when using a lactose induction system) or as a raw material for the steady-state growth of proteins during the latter half of logarithmic growth or at the start of steady-state growth.

[0124] To trigger a switch to lactose metabolism and lactose consumption (stopping the CCR), glucose levels are limited (glucose may even be depleted from the culture medium). Once this switch occurs, glucose can be added to the culture medium again to complete the sugar supply, or other sugars such as saccharose, fructose, or galactose can be added to the culture medium, especially in the form of molasses.

[0125] In one embodiment, whey is not present in the culture medium at the start of culture and is added when glucose levels are limited and a switch to lactose consumption (and induction of protein expression) is desired (during the logarithmic phase or at the start of the stationary phase).

[0126] In another embodiment, whey is added along with glucose at the start of culture and at the beginning of the logarithmic phase, but it is not consumed because the glucose concentration is maintained at a level high enough to induce CCR. As a result, the switch to lactose consumption is achieved by reducing the glucose supply for a predetermined time (30 minutes may be sufficient), and the bacteria then use the lactose present in the whey. A low amount of glucose may then be supplied again to prevent glucose accumulation in the medium (this can be confirmed by sampling the medium and measuring the glucose concentration). In general, stopping glucose supply to suppress CCR and then supplying glucose again later does not lead to the reappearance of CCR.

[0127] Therefore, when the microorganism is in the late logarithmic phase and / or stationary phase, whey is present in the culture medium. In one embodiment, when the microorganism is in the logarithmic phase, whey is present in the culture medium. In another embodiment, when the microorganism is in the logarithmic phase, whey is not present in the culture medium.

[0128] As described by others (Viitanen et al. 2003, Cheese whey-induced high-cell-density production of recombinant proteins in Escherichia coli, Microbial Cell Factories 2:2; de Diviitis et al. (2023) Cheese-whey permeate improves the fitness. Biotechnology for Biofuels and Bioproducts, volume 16:30), it is also possible to use sugars other than glucose, such as glycerol, to initiate the culture, in order to later add whey at an appropriate point in the culture (particularly to induce casein expression). The selection of sugars for adding whey and specific operating procedures can be readily designed by those skilled in the art.

[0129] In one embodiment, microbial culture is carried out in a fed-batch reactor by providing feed at regular intervals. In another embodiment, the culture is carried out in a continuous mode with continuous feeding and product withdrawal.

[0130] In another embodiment, the culture is performed in batch mode.

[0131] It should be noted that during fed-batch fermentation, there is controlled addition of substrates and supplements to the fermentation medium, in contrast to batch mode where all substrates are present in the bioreactor at the start of the method.

[0132] "Feed flow rate" represents the amount of culture medium, preferably containing microorganisms or bacterial host cells, that is transferred to the production reactor within a specific period of time, and may be expressed as volume per hour, for example, liters per minute. The feed flow rate may be regulated, for example, by an adjustable or controllable pump.

[0133] When a switch to lactose consumption occurs, it is preferable that the lactose concentration in the culture medium is higher than 1%, preferably 1% to 5% (by weight / volume, in the case of 1g / 100mL to 5g / 100mL).

[0134] In one embodiment, whey is unprocessed whey or whole whey, i.e., a liquid obtained from the coagulation or filtration of milk. This contains some whey proteins.

[0135] In another embodiment, the whey is obtained by ultrafiltration of the whole whey, and the permeate whey is obtained by removing the serum proteins from the whey.

[0136] In one embodiment, acidic whey (or permeate derived therefrom) obtained by coagulating milk via acidification by lactic acid bacteria is used. In this embodiment, a base is added to the culture medium to maintain the pH of the microorganism-containing solution at around pH 7.

[0137] In another embodiment, sweet whey is used.

[0138] In one embodiment, a composition containing casein and other proteins (as well as potentially microorganisms obtained from a microbial culture) is flowed from a first vessel through an apparatus including pipes or tubes, such tubular exchangers having both the function of moving the composition and heating it. Thus, there is heat transfer (or heat exchange) (corresponding to heating step iv) as the composition passes through the pipes so as to heat the composition, and such transfer occurs in at least a portion of the pipes. Such heating precipitates other proteins present in the solution, while the casein remains in the soluble fraction.

[0139] The soluble fraction may be recovered by centrifuging the liquid composition flowing from the pipe.

[0140] The concentration of casein, and / or the proportion of casein to other proteins, is higher in the recovered soluble fraction than in the initial composition, thereby enriching the soluble recovered fraction with casein.

[0141] As shown, composition i) contains a soluble fraction and an insoluble fraction. The soluble fraction represents the fraction that is not pelletized by centrifugation. The insoluble fraction is the pellet obtained after centrifugation. Centrifugation may be performed at approximately 3000 g for 20-30 minutes. On an industrial scale, the soluble fraction may be recovered using continuous flow centrifugation or other methods such as filtration. In the case of filtration, different types of membranes may be used.

[0142] The desired technical effect is obtained by heating while passing through the tube, and preferably in the absence of or without the addition of an organic solvent.

[0143] The apparatus may include a zone (such as a chamber or the length of a pipe) prior to the activated carbon zone for preheating the composition before it flows through the pipe to a desired temperature. For example, if the composition is at room temperature, preheating may raise the composition temperature to about 80°C. The apparatus may also provide a zone after the heating pipe zone for cooling the composition to a temperature below 100°C (preferably about 60 to a maximum of 80°C) so that the soluble fraction can be recovered.

[0144] When the relative amount of casein increases compared to the total amount of protein, the proportion of casein in the soluble fraction increases.

[0145] Other proteins are non-casein proteins, and therefore heating allows for the acquisition of a casein-enriched composition. The enrichment of casein in the composition represents an increase in casein in the soluble fraction compared to the amount present in the soluble fraction of the initial composition before flow through the pipe.

[0146] In one embodiment, the microbial composition is washed to remove the culture medium before heating in iv).

[0147] As an example, the cultured microbial cells (preferably bacteria) can be centrifuged, the pellet washed, and resuspended in a suitable liquid or fluid (a suitable buffer, more preferably water, and more preferably in the absence of organic solvents). The buffer is non-acidic (6.5 or higher), preferably near neutral (6.5 to 7.5), or basic (preferably less than 9). In fact, casein may precipitate at acidic pH. It is also preferable that the buffer is non-ionic or has low ionic strength.

[0148] Therefore, the resuspended composition is subjected to the heating step of iv.

[0149] In one embodiment, the microorganisms of the microbial composition are dissolved before iv) to obtain a liquid composition, which is in the first container. In another embodiment, the microorganisms in the microbial composition are not dissolved, and heating in iv) dissolves the microorganisms.

[0150] Microbial lysis can be carried out by any method known in the art, preferably using a chemical substance (such as a detergent), or by enzymatic lysis using, for example, lysozyme or proteinase K, or mechanically using a French press.

[0151] The soluble fraction recovered in v) may be subjected to one further step as needed, selected from the following: addition of activated carbon, chemical resin, membrane filtration (ultrafiltration, nanofiltration, or reverse osmosis), centrifugation, chromatography, or casein precipitation. The soluble fraction may be recovered, in particular, by centrifugation of the liquid composition flowing from the pipe, using a conventional centrifuge or a disk stacking centrifuge. Alternatively, it can be obtained by microfiltration or diafiltration.

[0152] Activated carbon can be added to the soluble fraction and stirred. The activated carbon can be used to adsorb impurities (particularly organic impurities or chlorine) present in the soluble fraction that were not removed when the soluble fraction was recovered in iii). Stirring with the activated carbon is preferable, and the activated carbon is removed before another method (such as those disclosed below) is performed on the soluble fraction.

[0153] Chromatographic methods are widely used in purification, including affinity chromatography, ion exchange chromatography, hydrophobic interaction chromatography, and others.

[0154] Membrane filtration, particularly ultrafiltration and nanofiltration, is commonly used in protein purification (Saxena et al. (2009) Membrane-based techniques for the separation and purification of proteins: An overview. Advances in colloids and Interface Science Volume 145, pages 1-22), and membrane filtration techniques are widely used in the dairy industry. Interestingly, these can be used to separate different caseins from each other (see above).

[0155] Furthermore, the specific properties of casein (Post et al. (2012) Effect of temperature and pH on the solubility of caseins: Environmental influences on the dissociation of caseins. J. Dairy Sci. Volume 95: pages 1603-1616), and in particular, their tendency to precipitate under acidic conditions, can be used for further purification.

[0156] In a preferred embodiment, the soluble fraction is further treated by casein precipitation under acidic conditions. After precipitation, the casein can be resuspended and resolubilized using a suitable basic buffer (Post et al. (2012) Effect of temperature and pH on the solubility of caseins: Environmental influences on the dissociation of caseins. J. Dairy Sci. Volume 95: pages 1603-1616). In a preferred embodiment, the soluble fraction is further treated by casein precipitation at a pH in the range of pH=4 to pH=5. In a more preferred embodiment, casein precipitation is carried out at a pH of about 4.6. Various acids, including lactic acid, hydrochloric acid, and sulfuric acid, can be used.

[0157] Next, the casein can be recovered by methods such as centrifugation or tangential flow filtration. A washing step may be added using water adjusted to the pH used for precipitation. The washed casein can be separated using the methods cited above.

[0158] Precipitation under acidic conditions is particularly interesting when isolating casein from microbial cultures, as it allows for the removal or degradation of any nucleic acids that may be present in the casein composition. When casein is used to obtain edible compositions intended for human consumption (such as cheese substitutes), the presence of microbial DNA or RNA may prove harmful (at least from a regulatory standpoint). The pH is approximately 4.6 and its range, the temperature is in the range of 80°C to 140°C, and the time can vary from a few minutes to two hours.

[0159] Alternatively, recombinant DNA can be removed using nucleases such as DNAse, which can be removed by heating.

[0160] When using microorganisms such as Gram-negative bacteria, additional steps may be taken to remove lipopolysaccharide (LPS), and high-temperature treatment under acidic or basic conditions, use of activated carbon at high temperatures, or filtration methods (10-20 kDa separation, or various extraction methods may be used).

[0161] Next, the pH can be neutralized to obtain the casein salt. For this purpose, various basic compounds such as liquid or gaseous ammonia, sodium hydroxide, potassium hydroxide, or calcium hydroxide can be added. The target pH may be in the range of 6.5 to 9. The kinetics of pH adjustment may be continuous or follow a series of plateaus to achieve a stable target pH. Before drying, the casein can be concentrated by filtration or evaporation. The casein can then be dried by spray drying, flash drying, or other methods known in the art.

[0162] In a preferred embodiment, the pH is adjusted using sodium hydroxide. In another embodiment, the pH is adjusted using potassium hydroxide. In yet another embodiment, the pH is adjusted using calcium hydroxide.

[0163] In certain embodiments, the casein composition is dried. When obtained from a bacterial culture, such a composition should contain about 15% (by weight) to 30% (by weight) of casein and carbohydrates in the dried composition.

[0164] It is preferable that a small amount of water remains to promote future rehydration. Therefore, drying should be understood as reducing the amount of water. In some embodiments, the amount of water is about 50% (by weight) or less.

[0165] The insoluble fraction, which contains the majority of the biomass produced during fermentation, can be processed separately and given added value. This biomass can be used for feed or food applications after being treated in the same way as casein to remove recombinant DNA: the pH is in the range of 4.6, the temperature is in the range of 80°C to 140°C, and the time can vary between a few minutes and 2 hours; or the recombinant DNA can be removed using a nuclease such as DNAse, which can be removed by heating.

[0166] Therefore, when the composition of i) in the method disclosed above passes through the pipe, it is heated to a temperature higher than 75°C (heating temperature). More preferably, the temperature is higher than about 80°C, more preferably higher than about 85°C, more preferably higher than about 90°C, more preferably higher than about 95°C, and most preferably higher than about 100°C or higher. It may be recommended to use temperatures of 105°C or higher, or 110°C or higher. Higher temperatures of 140°C or even 150°C are possible. It is preferable to heat the composition for a maximum of 3 minutes, or even more preferably a maximum of 1 minute. In this case, a suitable temperature is preferably 95°C to 115°C, preferably 100°C to 115°C (including the limit). A temperature of about 110°C would be suitable. Such temperatures allow for technical and functional effects of removing other proteins, thereby reducing their amounts and / or moving casein from the insoluble fraction to the soluble fraction, while heating for a short time (up to 5 minutes, preferably up to 2 minutes, but rather 90 seconds or less, or 75 seconds or less, more preferably 60 seconds or less, or even 45 seconds or less). Examples have shown that durations of 30 seconds or even 2 seconds can be applied. However, it is preferable to heat the composition flowing through the pipe for at least 5 seconds or at least 10 seconds.

[0167] The pressure inside the pipe is typically higher than 3 bar, more preferably 4 bar or more, and more preferably 4.5 bar or more. This is typically 7 bar or less, preferably 6.5 bar or less, and more preferably 6 bar or less. Pressures between 4.5 bar and 5.5 bar (including the limit) are well-suited.

[0168] When referring to measurable values, the term "about" means that a variation of ±3% from the specified value is included, where such variation is appropriate for performing the disclosed method.

[0169] The examples illustrate various conditions that enable the purification of casein from recombinant Escherichia coli (E. coli). While these examples also describe specific conditions, it is worth noting the specific temperature / holding time lengths at these temperatures (110°C / 75 sec; 110°C / 30 sec; 120°C / 30 sec; 140°C / 30 sec; 140°C / 2 sec) disclosed to address the technical problem of purifying casein. It is sometimes recommended to avoid overheating the composition and to use longer times rather than very short times. All of the various conditions (using heating temperatures from 95°C to 140°C and holding times from 2 seconds to 120 seconds in the heated chamber) enabled the acquisition of moderately concentrated casein with good purity in the soluble fraction after heat treatment.

[0170] It is recommended to preheat the composition coming from the first container before carrying out the heating process in the pipe. This is done in a chamber outside the first container, or in the pipe that guides the composition from the first container to the pipe to be heated for a desired time and at a desired temperature. The preheating temperature may be 20°C or 30°C lower than the target heating temperature, and may be about 20°C to about 60°C lower than the heating temperature. Preheating to 75°C to 90°C (e.g., 80°C) is well suitable. The total duration of the process (preheating + heating + chamber holding (maintaining at the heating temperature) + pre-cooling + cooling) was about 150 to 350 seconds, preferably 150 to 320 seconds, or 200 to 320 seconds.

[0171] To obtain the desired temperature within the pipe, especially when heating by heat exchange, heating is performed at a temperature higher than the desired temperature (usually about 5°C higher) to account for the temperature gradient from the outside of the pipe to the center of the pipe. Heating can be carried out by one of the following methods:

[0172] Direct steam injection: Steam is injected directly into the pipe to heat it. The steam is generated in a boiler and sent to the pipe, where it transfers heat to the pipe walls and heats the product flowing through it.

[0173] Indirect heating by heat exchange: Double-walled pipes are used. The composition flows through the inner tube, and the steam circulates in the space between the two walls. Heat from the steam is transferred through the pipe walls, heating the product.

[0174] Electric heating: A heating element is incorporated into the pipe to heat it, using electricity to generate heat directly on the pipe wall.

[0175] Inductive beam heating: This method uses electromagnetic induction to heat a pipe by the Joule effect, by using an alternating current induced in the pipe wall with the help of a magnetic field.

[0176] In some embodiments, the composition is circulated through the pipe at a flow rate of 15 L / hour to 30 L / hour.

[0177] The composition is usually cooled and, after flowing, supplied to a second container. In some embodiments, the soluble fraction, recovered by centrifugation and / or filtration after flowing and heating, is supplied to the second container. Further purification or concentration of casein (e.g., by evaporation) and / or quantification of casein in the soluble fraction can be performed.

[0178] This method makes it possible to obtain a casein composition in which casein accounts for at least 60%, preferably at least 62%, preferably at least 65%, preferably at least 67%, and preferably at least 70% of the total protein in the soluble fraction. In particular, the proportion of casein relative to total protein is increased in the soluble fraction compared to the proportion in the microbial composition.

[0179] In summary, the methods disclosed herein make it possible to obtain casein compositions with low environmental impact. Such casein compositions that can be obtained (or have been obtained) by the methods disclosed herein are further subject matter of the present invention. Such compositions can be characterized as defined above.

[0180] In milk, casein is present in the micelle form found in milk, and these micelles include alpha-S1 casein, alpha-S2 casein, beta-casein, and kappa-casein assembled within the same particle. In this invention, casein may not be assembled in the micelle form. However, this does not prevent the ability to obtain curd from the casein composition by adding a suitable coagulant.

[0181] In fact, as demonstrated in the examples, such casein compositions can be used to produce cheese substitutes, and in particular animal-free cheese substitutes, i.e., cheese substitutes that do not contain any animal-derived products (especially when the casein is isolated from a bacterial or yeast culture).

[0182] In summary, simply heating a composition containing casein and other proteins through a pipe for a short time (less than 5 minutes, or preferably less than 2 minutes) allows for enrichment of the casein in the composition by reducing the amount of the other proteins in the soluble fraction. Heating also allows for the solubilization of casein found in the insoluble fraction of bacterial cultures. The resulting casein, while not under optimal conditions for micelle formation, can be properly coagulated by the action of a coagulant. This finding was unexpected.

[0183] In particular, as shown in International Publication No. 2022 / 253816, despite the fact that recombinant casein does not have post-translational modifications (such as phosphorylation) and despite the fact that kappa-casein is known to play an important role in the formation of milk casein micelles, coagulation can be achieved using bacterial recombinant casein in the absence of kappa-casein.

[0184] One embodiment discloses a method for obtaining a dairy substitute, comprising: performing the method disclosed herein to enable obtaining a casein composition; mixing the casein composition with at least one other component, including at least one component selected from the group consisting of proteins, water, calcium, lipids, and carbohydrates, to obtain a liquid precurd composition (LpCC); and processing the liquid precurd composition (LpCC) to obtain a dairy substitute. Further processing of the LpCC includes, in particular, providing the LpCC with a coagulant to obtain curd. Such curd can then be drained (and the resulting whey may be reused in the method disclosed herein, thereby providing cyclical use), shaped, and aged to obtain a cheese substitute. A curd can be obtained and processed with lactic acid bacteria (Streptococcus thermophilus and Lactobacillus bulgaricus) to obtain a yogurt substitute.

[0185] Such embodiments are disclosed in particular in International Publication No. 2022 / 058573 and International Publication No. 2022 / 253816.

[0186] As indicated, the coagulant is preferably an acidifying agent, particularly lactic acid bacteria, or lactic acid, citric acid, or acetic acid, rather than rennet. The casein composition may contain only alpha-S1 casein (as casein protein), or only alpha-S2 casein (as casein protein), or only beta-casein (as casein protein), or only alpha-S1 casein and beta-casein (as casein protein), or only alpha-S2 casein and beta-casein (as casein protein), or only alpha-S1 casein and alpha-S2 casein (as casein protein), or alpha-S1 casein, alpha-S2 casein, and beta-casein.

[0187] The curd may be processed to obtain edible compositions, in particular cheese substitutes, as disclosed in International Publication No. 2022 / 058573 and International Publication No. 2022 / 253816. These two documents are incorporated herein by reference in part, in particular, in the parts describing obtaining LpCC or edible compositions starting from compositions containing casein, in particular bacterial casein, and / or casein without kappa-casein, in particular, in the parts relating to obtaining cheese substitutes having the essential characteristics of fresh cheese and cheese substitutes having the essential characteristics of soft or semi-soft cheese. The parts of these documents, in particular International Publication No. 2022 / 253816, relating to calcium and other salts, lipids, emulsifiers and gelling agents, carbohydrates, vitamins, coagulants, fermented products, moisture, and processing of the curd are also incorporated herein by reference.

[0188] Depending on the desired type of cheese substitute, the teachings regarding the composition of LpCC, as well as the amount of gelling agent to be added (particularly pp. 24-28 of International Publication No. 2022 / 253816), are also incorporated by reference.

[0189] It is conceivable that a gelling agent (especially agar) can be added along with other components before or after solidification.

[0190] b) is preferable if all other components added are of non-animal origin.

[0191] The components added in ii) are i. Proteins other than casein as needed ii. Lipids iii. Water, and iv. Carbohydrates It includes; in a preferred embodiment, the carbohydrates are lactose-free so that the edible composition is more acceptable to customers with lactose intolerance.

[0192] The selection and quantity of ingredients are adjusted to ensure that the composition of LpCC conforms to the desired final composition for the edible composition, taking into account water loss due to coagulation and maturation (which can be controlled by those skilled in the art by changing the duration and conditions of maturation).

[0193] In particular, the LpCC processing is vi. Adding at least one coagulant to the liquid composition in order to obtain a card, vii. Further processing the card to obtain a dairy substitute, wherein the dairy substitute is a cheese substitute or a yogurt substitute, Includes.

[0194] Such further step vi) may include acid precipitation of casein. In particular, the acid precipitation may be carried out at a pH of 4-5 (e.g., pH=4.6) at about 90°C, but preferably at room temperature (about 20°C). Several other purification steps can be performed.

[0195] Further processing of the card is a) A step of mixing a structural agent selected from the group consisting of gelling agents, texturers, emulsifiers, and mixtures thereof with the card to obtain a complementary card, b) A step of forming and draining the completed card, It may include.

[0196] Furthermore, a) Prepare a casein composition comprising recombinant casein disclosed herein, b) Mixing the casein composition with at least one other component, which includes at least one component selected from the group consisting of water, calcium, lipids, and carbohydrates, to obtain a liquid precard composition (LpCC). c) Adding at least one coagulant to the liquid pre-card composition in order to obtain a card, d) Further processing the curd (particularly by draining) to obtain an edible composition, A method including, In b) and / or d), a gelling agent (especially agar) is added. I can list some methods.

[0197] In one embodiment, a gelling agent is added in step b).

[0198] In another embodiment, a gelling agent is added in d).

[0199] In another embodiment, a gelling agent is added in b) and d).

[0200] In this embodiment, the edible composition is i. Having a protein content in the range of 2-15% (by weight), ii. Fat-free with a water content of over 80% The case is preferable.

[0201] Therefore, this embodiment is well suited to the production of fresh cheese substitutes. Further processing of the curd may further include shaping the drained curd and / or ripening the dried curd.

[0202] The environmental impact of dairy substitutes, or other dairy substitutes made from casein obtained by the methods described herein, can be significantly reduced in at least one of the above criteria compared to dairy substitutes obtained by the same methods but without the addition of whey to produce casein by fermentation, where the lactose derived from whey is provided as is or replaced with an equal amount of glucose or sucrose provided in sucrose molasses. In particular, the environmental impact can be reduced by at least 5%, or at least 10%, or at least 20%, or at least 25%, or at least 30% for at least one of the above factors (especially climate change). [Brief explanation of the drawing]

[0203] [Figure 1] Structure of a plasmid expressing beta-casein and alpha-S1 casein. [Figure 2]SDS-PAGE analysis of αs1-casein and β-casein in soluble and insoluble fractions of the dissolved product, before and after heat treatment. Lane 1: Size marker, Precision Plus Protein® Unstained Standards (Bio-Rad, 1610363EDU) - Lane 2: Cell suspension before heat treatment, soluble fraction - Lane 3: Lysate after heat treatment (Test 1), soluble fraction - Lane 4: Lysate after heat treatment (Test 2), soluble fraction - Lane 5: Lysate after heat treatment (Test 3), soluble fraction - Lane 6: Lysate after heat treatment (Test 4), soluble fraction - Lane 7: Lysate after heat treatment (Test 5), soluble fraction - Lane 8: Cell suspension before heat treatment, insoluble fraction - Lane 9: Lysate after heat treatment (Test 1), insoluble fraction - Lane 10: Lysate after heat treatment (Test 2), insoluble fraction - Lane 11: Lysate after heat treatment (Test 3), insoluble fraction - Lane 12: Lysate after heat treatment (Test 4), insoluble fraction - Lane 13: Lysate after heat treatment (Test 5), insoluble fraction. [Figure 3] SDS-PAGE analysis of αs1-casein and β-casein in soluble and insoluble fractions of lysates before and after heat treatment. Analysis of 3-fold diluted samples. For each panel, Lane 1: Size marker, Precision Plus Protein® Unstained Standards (Bio-Rad, 1610363EDU) - Lane 2: Cell suspension before heat treatment, soluble fraction - Lane 3: Cell suspension before heat treatment, insoluble fraction - Lane 4: Lysate after heat treatment, soluble fraction - Lane 5: Lysate after heat treatment, insoluble fraction. Panel A: 110°C, 75 sec; B: 110°C, 30 sec; C: 120°C, 30 sec; D: 140°C, 30 sec; E: 140°C, 2 sec. [Figure 4] Monitoring of growth and glucose concentration in culture medium under various culture conditions. Growth was monitored by optical density (OD) at 600 nm (A). Uninducible cultures in glucose are represented by dotted lines, cultures with IPTG-induced glucose by dashed lines, and cultures with glucose and whey by solid lines. [Figure 5]Analysis of casein production by SDS-PAGE under various induction conditions. Samples were prepared as described in Example 4. T1, T2, T3: Total lysates from uninducible culture in glucose (T1), culture with IPTG-induced glucose (T2), and culture containing glucose and whey (T3, as described in Example 4). P1, P2, P3: Insoluble fractions (pellets) of lysates from uninducible culture in glucose (P1), culture with IPTG-induced glucose (P2), and culture containing glucose and whey (P3). S1, S2, S3: Soluble fractions (supernatant) of lysates from uninducible culture in glucose (S1), culture with IPTG-induced glucose (S2), and culture containing glucose and whey (S3). A: Purified alpha-S1 casein and alpha-S21 casein from Sigma, 1 g / L. A: Purified beta-casein from Sigma, 1 g / L. M: Molecular weight marker. The band size is shown on the left side of the gel. [Figure 6] A comparison of the environmental impacts of casein production using different raw materials. The use of glucose is presented as the reference standard (100%). [Figure 7] SDS-PAGE of clarified cell lysates from the sample; (1) BR1-SO_gal0, (2) BR2-SO_gal1, (3) BR3-SO_gal2, (4) BR4-SO_gal3 (without whey), and (5) BR5-SO_gal3 (with whey). For control, commercially available alpha-casein (A) and beta-casein (B) solutions. [Figure 8] SDS-PAGE of insoluble fractions of lysates from the samples: (1) BR1-SO_gal0, (2) BR2-SO_gal1, (3) BR3-SO_gal2, (4) BR4-SO_gal3 (without whey), and (5) BR5-SO_gal3 (with whey). For control, commercially available alpha-casein (A) and beta-casein (B) solutions. [Figure 9] Concentrations of lactose (A), glucose (B), and galactose (C) in the culture medium. [Examples]

[0204] Example 1: Production of alpha-S casein and beta-casein Synthetic genes encoding alpha-S1 casein and beta-casein (related to the native genes P02662 and P02666, respectively) were modified to remove the signal peptide and optimize codon use for expression in Escherichia coli. The sequences of the proteins encoded by the novel synthetic open reading frames (SEQ ID NOs. 2 and 4) and the sequences of the novel synthetic open reading frames are shown in the last column of Table 2. The two synthetic open reading frames were cloned together into pET25b+ within the same operon under the control of the T7 promoter, replacing the NdeI-HindIII small fragment of this vector, so that (i) both synthetic open reading frames were under the control of the T7 ribosome binding site and terminated by two TAA stop codons, and (ii) the beta-casein open reading frame was close to the promoter and the alpha-S1 open reading frame was distal to the promoter (Figure 1). The resulting plasmids were transformed into BL21(DE3) strain (Novagene). Individual transformed clones were isolated, and each synthetic gene was identified. One clone was used for inoculation into LB medium.

[0205] [Table 2]

[0206] To produce batches of alpha-S1 casein and beta-casein, the obtained strains were cultured as follows:

[0207] Five concentrated stocks of each strain (1 mL each, stored in 10% DMSO at -80°C) were thawed and used to inoculate five pre-cultures of 1 L each in reactivated medium Y15 consisting of yeast extract (15 g / L) and NaCl (5 g / L), supplemented with 100 μg / mL ampicillin (inoculation rate of 0.1% (volume / volume)). The five pre-cultures were grown in 4 L or 5 L Erlenmeyer flasks at 30°C for 9 hours at 170 rpm (swirl diameter: 25 mm). After 9 hours, an optical density in the range of 3 (60 nm) was achieved.

[0208] Using 2.4 L of the initial pre-culture, it was inoculated to a volume of 120 L in optimized culture medium supplemented with 100 μg / mL ampicillin and 15 g / L glucose (inoculation rate of 2% (volume / volume)). Growth parameters (oxygen pressure, agitation, pressure, aeration rate, and pH adjustment) were optimized to achieve an optical density (600 nm) in the range of 15.

[0209] Using 90 L of this culture, it was inoculated into a 1500 L initial volume fermenter in optimized culture medium supplemented with 100 μg / mL ampicillin (inoculation rate of 6% (volume / volume)). Growth parameters (glucose feed, oxygen pressure, agitation, pressure, aeration rate, and pH adjustment) were optimized to achieve an optical density in the range of 90 (600 nm). When the optical density reached 20, IPTG was added at a final concentration of 0.2 mM. This yielded approximately 2 tons of culture medium with 3.4% dry biomass.

[0210] Example 2: Thermal dissolution of bacteria by ultra-high temperature (UHT) treatment At extremely high temperatures, α s1 We established protocols for the extraction and solubilization of casein and β-casein.

[0211] Biomass concentration and washing The culture medium obtained as described in Example 1 was concentrated using a self-cleaning disk-type centrifuge (centrifugal force: 20,000 G, temperature < 20°C). 298 kg of concentrated cells were recovered.

[0212] Next, the concentrated biomass was washed three times with leachate using the same centrifuge. The concentrated biomass was then mixed with water until a homogeneous suspension was obtained. The suspension was then separated by centrifugation to remove the first fraction of the washing water. This washing procedure was repeated twice, and finally, a pellet of concentrated cells weighing 239.1 kg was obtained.

[0213] Thermal dissolution of bacteria Next, the washed concentrated biomass was resuspended in 4.2 times its volume of leachate until a homogeneous suspension was obtained. 60 L of this suspension was stored at +4°C. The suspension was then divided into four 10 kg fractions and one 8.15 kg fraction. The pH of each suspension was adjusted to 8.5 using a 2 M sodium hydroxide (NaOH) solution.

[0214] Next, five fractions were heated using a UHT sterilization device (sterilization device, OMVE, HTST / UHT system, HT220-DSI, configuration: tubular heat exchanger, flow rate in chamber: 16.66~27.2 L / hour, holding time (time in chamber): 2~75 seconds) by monitoring the holding time at a predetermined temperature in the sterilization chamber. The temperature in the sterilization chamber was 110~140°C.

[0215] To determine the optimal and rapid heat treatment that would enable efficient bacterial lysis, E. coli protein precipitation, and casein solubilization, five different conditions were tested on five bacterial suspensions. The different set heating temperatures were slightly higher than the temperatures applied in the sterilization chamber to ensure that these final temperatures were achieved. Test 1: Temperature = 110°C, Holding time = 75 seconds Test 2: Temperature = 110°C, Holding time = 30 seconds Test 3: Temperature = 120°C, Holding time = 30 seconds Test 4: Temperature = 140°C, Holding time = 30 seconds Test 5: Temperature = 140°C, holding time = 2 seconds, on a micropilot scale.

[0216] In each case, the product temperature was controlled during the heating process and at the outlet of the sterilization chamber, while the supply pressure was measured throughout the entire process.

[0217] To investigate the efficiency of these five different heat treatments, samples were collected before and after each test and analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) on a polyacrylamide gel (4-20% Criterion® TGX Stain-Free Protein Gel, Bio-Rad, 5678093) using Precision Plus Protein® Unstained Standards (Bio-Rad, 1610363EDU) as the ladder.

[0218] For each test, 1 mL of bacterial suspension was taken before and after the applied heat treatment and centrifuged (13000 rpm, 5 minutes, room temperature) to separate the soluble and insoluble fractions. The supernatant was separated from the pellet and mixed in a 1:1 ratio with a double sample buffer consisting of 0.1 M Tris-HCl, 4% vol:vol SDS, 0.2% wt:vol bromophenol blue, and 20% vol:vol glycerol. The cell pellet was mixed with 1 mL of 2% vol:vol SDS solution until a homogeneous suspension was obtained, in the same ratio as the double sample buffer described above. Each resulting solution was heated at 95°C for 5 minutes and loaded into the wells of a polyacrylamide gel using a running buffer consisting of Tris-HCl (25 mM), glycine (250 mM), and SDS (0.1% vol:vol). Next, the gels were visualized under UV light in a Gel-Doc® EZ Imager (Bio-Rad). Diluted samples (3x) were also used to achieve better quantification. The ratio of casein content to total protein content was determined by analyzing each SDS-PAGE gel using Imagelab software, comparing the band intensity of casein with the band intensity of other proteins from Escherichia coli (E. coli).

[0219] The results are summarized in Figures 2 and 3, showing undiluted (Figure 2) and diluted (Figure 3) samples. For all test conditions for sterilization, Escherichia coli (E. coli) cells were efficiently lysed, and α s1 most of the casein and β-casein were solubilized: The total amount of casein was very similar before and after sterilization, but these caseins were essentially in the insoluble fraction before sterilization (Figure 3, lane 8) and in the soluble fraction after sterilization (Figure 3, lanes 3 - 7), indicating that the yield of the method is very high (at least >80%).

[0220] The ratio of casein to total protein in the soluble fraction of the lysate (Figure 3, lane 4 of each panel) was significantly the same in five tests and was in the range of 75% - 77%. This ratio was found to be significantly higher than the ratio in the insoluble fraction of the pre-lysis cells (Figure 3, lane 3 of each panel) which showed many additional protein bands as seen in Figure 3 (lane 3 of each panel). Naturally, this ratio is higher than the ratio in the pre-lysis cell composition (insoluble + soluble), indicating that the soluble fraction of the lysate is enriched in casein compared to the initial composition.

[0221] In addition, the average α s1 -casein / β-casein ratio was also very similar among the five tests, in the range of 47% - 53%, similar to the initial composition in the pre-treatment cells, indicating that there was no bias in the purification of α s1 -casein versus β-casein under the conditions used by the inventors.

[0222] Example 3: Thermal lysis of bacteria As described in International Publication No. 2022 / 253816, synthetic genes encoding alpha-S1 casein, alpha-S2 casein, and beta-casein (corresponding to native genes P02662, P02663, and P02666, respectively) were modified to remove the signal peptide. These were cloned into plasmids, and the resulting plasmids were transformed into the BL21(DE3) strain. Individual transformed clones were isolated, and for each synthetic gene, one clone was used to inoculate into LB medium.

[0223] Cells were lysed using two different protocols, and samples from the total extract, as well as the soluble and insoluble fractions, were analyzed by SDS-PAGE. Casein was monitored in the soluble and insoluble fractions of the cell extract using two different protocols. Cells transformed with blank vectors were used as controls.

[0224] Protocol 1: A cell pellet obtained from 100 mL of culture was 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 minutes, followed by sonication for 10 seconds (amplitude 10%, Q Sonica XL-2000). 10 μL of the total fraction was collected and stored for SDS-PAGE analysis. The soluble and insoluble fractions were separated by centrifugation at 3220 g at 4°C for 20 minutes. The supernatant was collected and supplemented with 10% glycerol for storage. The pellet was further resuspended in 50 mM Tris containing 2% SDS, and supplemented with 10% glycerol for storage. 10 μL of each fraction was collected for SDS-PAGE.

[0225] Protocol 2: A cell pellet obtained from 100 mL of culture was lysed by resuspension in Bugbuster (Millipore) containing 0.4% Lysonase (Millipore). The mixture was incubated at room temperature for 5 minutes, then centrifuged at 3220 g at 4°C for 20 minutes. The supernatant was collected, and 10 μL of the supernatant was taken as a reserve for SDS-PAGE analysis. The pellet was further resuspended in 2% SDS, and then 10 μL of the suspension, representing the insoluble fraction, was taken and saved for SDS-PAGE analysis.

[0226] As is often observed in the overexpression of recombinant proteins in Escherichia coli (E. coli), alpha-S1 casein was found in both the soluble (S) and insoluble (P) fractions, while alpha-S2 casein was found primarily in the insoluble fraction, and beta-casein was found mostly (protocol 1) or completely (protocol 2) in the insoluble fraction. This suggests that casein can be present in inclusion bodies in Escherichia coli (E. coli). The differing quantitative results may be due to differences in the solubility of beta-casein or differences in the stability of the inclusion bodies depending on the protocol. Nevertheless, recovery of casein from the soluble fraction of the extract using these protocols will result in the loss of most or all of the recombinant protein.

[0227] The effect of heat-induced dissolution on casein was tested using the recombinant bacterial strains described above. Clones transformed with empty vectors were used as controls.

[0228] The culture was centrifuged, the cell pellet was resuspended in 1:1 volume of sterile water, centrifuged again, washed with another 1:1 volume of water, and resuspended in 1:1 volume of water. A 1 mL sample of this cell suspension was treated by heating at 95°C for 0 (unheated), 10, 20, 30, 60, 90, and 120 minutes to induce cell lysis, and the 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 were approximately the same amount as the total cell extract. Without heating, cell lysis did not occur, or only residual cell lysis occurred in water. The pellet essentially contained whole cells including casein, and this sample is indeed a whole cell extract.

[0229] It was confirmed that dissolution due to temperature affects the distribution of casein in the soluble and insoluble fractions.

[0230] Before heating at 95°C for 10 minutes (or earlier), alpha-S1 casein was found almost entirely in the soluble fraction, but without heating, it was found to an equal degree in both the soluble and insoluble fractions.

[0231] Beta-casein progressively migrated to the soluble fraction, containing a large amount of this recombinant protein by 10 minutes of heating (or earlier), and the major portion by 30 minutes. Without heating, beta-casein was found almost entirely or completely in the insoluble fraction.

[0232] The distribution of alpha-S2 casein (which is entirely insoluble without heating) appears to be less heat-sensitive, as it was still largely found in the insoluble fraction even after 120 minutes of heating. Nevertheless, it appeared in the soluble fraction after 20 minutes of heating at 95°C, and its presence in the insoluble fraction decreased slightly over time.

[0233] Many other protein bands originating from the insoluble fraction also decreased over time, but they did not transfer to the soluble fraction. In the soluble fraction of the heated sample, only a few faint protein bands were observed in addition to the casein band.

[0234] These results indicate that dissolution by heating is an excellent method for the rapid purification of casein, particularly alpha-S1 casein and beta-casein.

[0235] Example 4: Production of recombinant casein in a 1L fermenter Acidic whey was used as a raw material in combination with glucose. To produce acidic whey, semi-skimmed pasterised milk was incubated with 0.1% lactic acid bacteria (Choozit MBT LYO 20 DCU) at 34°C for approximately 8 hours until the pH reached approximately 4.6, inducing coagulation. The curd was drained for 48 hours to collect the whey, yielding 0.77 kg of whey per liter of milk, with a pH of 4.6 and a lactose concentration of 51.3 g / L. This whey was then filtered using a 0.22 mM filter and autoclaved. SDS-PAGE analysis showed no residual casein in the whey.

[0236] The cultures of the recombinant strains described above were inoculated into 1 L fermenters (Multifors 2 Microbila 1.4 L TV, Infers) in a medium containing whey and glucose as carbohydrate sources.

[0237] A culture medium supplemented with 50% whey was used. This medium was also supplemented with 100 μg / ml ampicillin and 2 g / L glucose. The lactose concentration in this medium was approximately 25.7 g / L.

[0238] In 0.5 L of culture medium, the recombinant clone preculture was inoculated, and the initial OD (Oral Dissociation) was performed. 600 The ratio was set to 0.25. Fermentation was carried out in fed-batch mode, with pH=7.2; T=37℃; pO2=15%, and the culture was supplied with a solution containing 600 g / L of D-glucose. After 14 hours, ampicillin was added (100 μg / ml in the culture).

[0239] Lactose can induce casein expression from the pET vector at BL21. However, at the start of the run, glucose could suppress lactose uptake through carbon catabolite repression (Gorke, B., Stulke, J. Carbon catabolite repression in bacteria: many ways to make the most out of nutrients. Nat Rev Microbiol 6, 613-624 (2008); Postma PW, Lengeler JW, Jacobson GR. Phosphoenolpyruvate:carbohydrate phosphotransferase systems of bacteria. Microbiol Rev. 1993 Sep;57(3):543-94). Therefore, at the start of the run, only glucose is consumed.

[0240] After about 14 hours, OD 600At 40°C (Figure 4), glucose supply was interrupted for 30 minutes, resulting in a rapid decrease in glucose concentration in the culture medium. This enabled a metabolic switch that allowed lactose consumption, inducing casein expression. After 30 minutes, glucose supply was resumed, but reduced to allow for culture growth without hindering lactose consumption. In this respect, the induction process differed from that used by Viitanen et al. (Viitanen et al. 2003, Cheese whey-induced high-cell-density production of recombinant proteins in Escherichia coli, Microbial Cell Factories 2:2); in this study, induction was achieved by adding whey to a medium that did not contain glucose but was supplemented with glycerol. The culture was maintained for T=21 hours, OD 600 The process stopped at 100.7. No lactose was detected in the culture medium by this point. Cells were harvested by centrifugation at room temperature.

[0241] As a negative control, the same medium was used, but without whey (substituted with water), while maintaining the same final concentrations for other components. Therefore, glucose is the sole carbohydrate source, and casein expression is not expected. The culture was performed for T=21 hours, OD 600 The cell culture was stopped at OD=94.6. Cells were harvested by centrifugation at room temperature. As a positive control for casein expression, induction was achieved using the same medium without whey (replaced with water) at OD=40 by adding isopropyl β-D-1-thiogalactopyranoside (IPTG), a lactose analog whose inductive effect is not inhibited by glucose. The culture was maintained for T=21 hours, OD 600 The process was stopped at 80.0. Cells were harvested by centrifugation at room temperature. The conditions are summarized in Table 3.

[0242] [Table 3]

[0243] For analysis, cell aliquots were centrifuged and resuspended in half the volume of sterile water. A 1 mL sample of this cell suspension was treated by heating at 95°C for 60 minutes, as described above (European PCT Patent Application No. 2022 / 064728), resulting in cell lysis, solubilization of casein, and degradation and / or precipitation of other proteins in the soluble fraction, and the soluble and insoluble fractions were separated by centrifugation. The insoluble fraction was resuspended in 1 mL of 2% SDS solution, and 10 μL aliquots of both the soluble and insoluble fractions were analyzed by SDS-PAGE along with the total lysate.

[0244] Purified beta-casein (Sigma) derived from milk was used as a control, but it exhibited a higher apparent molecular weight, 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).

[0245] As shown in Figure 5, casein expression was efficiently induced by both IPTG and whey-containing media in the presence of glucose. Casein production was very efficient using whey. Recombinant casein production ranged from approximately 2.5 g / L with glucose to 5 g / L with whey, when normalized by cell density (see OD above). 600 (Reference) It was estimated to be twice as high and 1x higher.

[0246] The amount of sugar consumed was estimated for all samples. Under the conditions used, *E. coli* BL21 is capable of taking up and metabolizing glucose. At low glucose concentrations, lactose can also be taken up and hydrolyzed to glucose and galactose; however, galactose is hardly consumed, while glucose derived from lactose is metabolized very efficiently. Therefore, glucose consumption was estimated by determining the difference between the glucose added to the culture medium and the glucose produced from the hydrolysis of lactose. Given that the glucose concentration in the culture medium was negligible at the end of the experiment, the estimation was based on the mass of added sugar.

[0247] The results are shown in Table 4. As shown in this table, both added glucose and glucose from lactose hydrolysis were efficiently consumed during the fermentation run, but the consumption of glucose from whey (from lactose) was very low until the glucose concentration decreased. Under the conditions used, glucose from lactose hydrolysis accounted for 12% of the total glucose consumption, indicating that lactose contributed to the carbohydrate source. Galactose from lactose hydrolysis was not consumed.

[0248] [Table 4]

[0249] Example 5: Environmental impact The environmental impact of casein obtained by bacterial fermentation using various sugars as raw materials can be quantified using a multi-criterion life cycle assessment (LCA) approach. This study was conducted according to the European EF 3.1 method (https: / / eplca.jrc.ec.europa.eu / LCDN / developerEF.xhtml).

[0250] The life cycle inventory (LCI) is based on average primary data from laboratory and pilot-scale experiments. Data that was not collected directly from experimental results is based on industrial predictions from manufacturing methods, particularly regarding utilities and processes, or predictions from its partners.

[0251] The effects of various sugar raw materials were tested. Glucose was considered to be obtained from corn and its origin was assumed to be Europe. Sucrose was considered to be obtained from sugar beets and its origin was assumed to be the rest of the world (worldwide except Switzerland). Molasses was considered to be obtained from sugar beets, its origin was assumed to be the rest of the world, and it was assumed to contain 50% sugar by weight as sugar content.

[0252] Whey was considered to be obtained from the cheese manufacturing process and its origin was assumed to be worldwide.

[0253] The differences were only in the environmental impact of the raw materials, and it was assumed that changing the raw materials did not affect other parameters.

[0254] The LCI of each sugar raw material was calculated using the Ecoinvent 3.9 database according to its origin.

[0255] The differences compared to Example 4 are as follows: - The strain was gal+, i.e., having a proficient Leloir pathway and capable of consuming the galactose portion of lactose. - When using sucrose or molasses, the strain was assumed to be able to consume sucrose as described above in the text. - The sugar supplied to the strain was assumed to contain 100% glucose, or 68.6% glucose and 31.4% of another sugar (which could be sucrose from molasses or lactose from acid whey). - The fermenter was 350 L. - A portion of the water was reused in the process. The purification process included the following steps: -The biomass is collected as described in Example 2 and washed three times. - Heat the washed biomass at 95°C for 1 hour. -Collect the supernatant. -Precipitate the casein at pH 4.6 using HCl, and wash the precipitate. - Resuspend the casein pellet and neutralize it with sodium hydroxide (pH=7). - Dry the sodium caseinate by spray drying to reduce its moisture content to 5%.

[0256] A life cycle impact assessment (LCIA) was conducted using datasets from the Ecoinvent 3.9 database (Zurich, Switzerland) and Agrybalise (ADEME, France). All impacts were assigned to the casein composition and not to by-products of methods such as biomass. Acidic whey used as a carbon source can be considered either as waste or as a high-value by-product from soft cheese production. Therefore, in the former case, there is no environmental impact associated with the use of whey. In the latter case, it contributes to the emission of the final product according to dataset EF 3.1 from ecoinvent 3.9.

[0257] The results for the 16 criteria (see above for definitions of units) are shown in Figure 6 as relative values ​​compared to glucose as the sole raw material.

[0258] Regarding acidic whey as waste, its environmental impact is significantly smaller than that of glucose in all 16 criteria, according to the criteria: • Criterion ix (Toxicity to humans: Non-carcinogenic): Because sugar beet derivatives have the property of fixing heavy metals during the sucrose manufacturing process, compared to molasses, Criteria v, ix, and to a lower degree, criterion iii: compared to sucrose, With the exception of one factor, the effect is smaller or equivalent to that of other sugars in all other criteria.

[0259] For high-value-added acidic whey, which involves significant environmental impact, it corresponds to extensive land use, and in this case, the contribution of animals that affect the raw materials means that the environmental impact is greater than that of other sugars in terms of land use. However, this is: • Lower than glucose in all other criteria. • It is lower than sucrose in 11 of the 16 criteria (i, ii, iv, vi, vii, x, xii, xiii, xiv, xv, xvi). • It is lower than molasses in 8 of the 16 criteria (ii, v, vi, vii, x, xiv, xv, xvi).

[0260] In summary, using whey as a carbon source in methods for obtaining recombinant casein from microbial sources generally has a better environmental impact than the impact measured when using other carbon sources, based on a standard of 50% or more.

[0261] Example 6: Strain construction for lactose and galactose consumption The E. coli (E. coli) BL21(DE3) strain can consume glucose and partially lactose (but not galactose produced from lactose). A new E. coli (E. coli) BL21(DE3) strain was generated to express the galETKM operon derived from the E. coli (E. coli) K12 strain, which is necessary for galactose consumption. It should be recalled that the galETKM operon contains four genes; galE encodes epimerase, galT encodes uridilyltransferase, galK encodes galactokinase, and galM promotes the interconversion between β-galactose and α-galactose.

[0262] The constructed BL21(DE3) strain harbors the gal operon under different promoters, either on low copy number plasmids (pSO148 and pSO149) or within the chromosome (see sequences, Table 5).

[0263] [Table 5-1]

Table 5-2

Table 5-3

Table 5-4

Table 5-5

Table 5-6

Table 5-7

Table 5-8

Table 5-9

Table 5-10

Table 5-11

Table 5-12

Table 5-13

Table 5-14

Table 5-15

[0264] Example 7: Production of Recombinant Casein in a 1-L Fermenter Using Lactose- and Galactose-Consuming Strains Casein production using the BL21 strain, which can consume lactose and galactose, was tested using whey as a raw material in combination with glucose. Acidic whey was prepared as described in Example 4 and then centrifuged at 12,000 rpm at 4°C for 20 minutes. The supernatant was then filtered through a 0.22 μM filter.

[0265] Cultures of each recombinant strain listed in Table 6 were inoculated into 1 L fermenters (Multifors 2 Microbila 1.4 L TV, Infors) in a medium containing whey and glucose as the sole carbohydrate source.

[0266] [Table 6]

[0267] A medium (MSA) containing 50% whey was used, supplemented with 0.71 g / L Na2SO4, 11.3 g / L (NH4)2SO4, 0.85 g / L KH2PO4, 1.11 g / L Na2HPO4, 4 g / L MgSO4(7H2O), 1.6 mL / L 5% antifoaming agent solution (Struktol), and trace elements. The medium was sterilized by filtration through a 0.22 μM filter and supplemented with 100 μg / ml ampicillin and 33 g / L glucose. Spectinomycin was added to cultures using SO_gal1 and SO_gal2 at a final concentration of 100 μg / L of culture. The lactose concentration in this medium was approximately 20 g / L. For culturing using SO_gal3, a minimal medium was used (containing trace elements including 0.71 g / L Na2SO4, 11.3 g / L (NH4)2SO4, 0.85 g / L KH2PO4, 1.11 g / L Na2HPO4, 4 g / L MgSO4(7H2O), 1.6 mL / L 5% antifoaming agent solution (Struktol), and 20 g / L lactose). The conditions are summarized in Table 7.

[0268] [Table 7]

[0269] In 0.5 L of culture medium, the recombinant clone preculture was inoculated, and the initial OD (Oral Dissociation) was performed. 600nm The ratio was set to 0.25. Fermentation was carried out in fed-batch mode, with pH=7.2; T=37℃; pO2=15%. The culture was supplied with a solution containing 600 g / L of D-glucose. After 12.5 hours, ampicillin was added (100 μg / ml in the culture).

[0270] During the growth phase (t=0 to t=10 hours), glucose inhibits lactose uptake by suppressing carbon catabolism, and only glucose is consumed. Under our culture conditions, glucose was completely consumed after 10-12 hours, and no additional glucose was added for at least 30 minutes to allow the metabolic switch to lactose consumption and induce casein expression. The switch correlated with a spike in pO2 concentration in the bioreactor. The culture was stopped at T=17.8 hours, and OD 600 The range was 94-107.

[0271] For analysis, cell aliquots were centrifuged and resuspended in 1:1 aliquots of lysis buffer (Tris HCl, pH 8.5). A 2 mL sample of this cell suspension was treated by heating at 95°C for 60 minutes as described in Example 4. The soluble and insoluble fractions were separated by centrifugation. The insoluble fraction was resuspended in 1 mL of 2% SDS solution, and 10 μL aliquots of both the soluble and insoluble fractions were analyzed by SDS-PAGE. Casein expression was efficiently induced under all conditions, as shown in Figures 7 and 8. Recombinant casein production was estimated to occur in bacterial cultures ranging from 2.5 g / L to 6 g / L.

[0272] The consumption of lactose, glucose, and galactose was measured in all samples. The results are shown in Figure 8. As shown in these figures, lactose was completely hydrolyzed, and both the added glucose and the glucose resulting from lactose hydrolysis were efficiently consumed during the fermentation run. The galactose obtained from lactose hydrolysis was consumed in BR2, 3, 4, and 5 and accumulated in BR1. As expected, strains SO_gal1, SO_gal2, and SO_gal3 were able to consume galactose in contrast to SO_gal0.

Claims

1. A method for obtaining a casein composition, i. Prepare a microorganism transformed with at least one nucleic acid encoding casein, ii. Culturing the microorganism using a medium containing whey so as to express and produce casein, iii. This provides a microbial composition in which the pH of the composition is 6.5 or higher, preferably less than 9, iv. Heating the microbial composition to reduce the amount of other proteins in the soluble fraction of the composition, wherein the heating is carried out at a temperature of 75°C or higher. v. The soluble fraction is recovered from the heated cell composition of iv) This allows us to obtain a casein composition in the soluble fraction, Methods that include...

2. The method according to claim 1, wherein whey is present in the culture medium at the start of the culture.

3. The method according to claim 1 or 2, wherein whey is added to the culture medium during the logarithmic phase or the stationary phase.

4. The method according to any one of claims 1 to 3, wherein in ii, in addition to whey, another carbon source is present in the culture medium, and this other source of the medium is preferably a sugar, particularly glucose, fructose, saccharose, and optionally present in molasses.

5. The method according to any one of claims 1 to 4, wherein the microorganism is a bacterium.

6. The method according to claim 5, wherein the bacterium is Escherichia coli (E. coli), particularly gal+ Escherichia coli (E. coli).

7. The method according to any one of claims 1 to 6, wherein the expression of at least one nucleic acid is induced by the presence of lactose.

8. The method according to claim 7, wherein the at least one nucleic acid is under the control of a promoter fused to the laco operator region of a lactose operon, and the microorganism expresses the lacI repressor of the operon.

9. The method according to any one of claims 1 to 8, wherein the culture of the microorganism is started in the presence of a sugar other than lactose, and the consumption of the lactose present in the whey is started during the logarithmic growth phase, particularly around the mid-phase of the logarithmic growth phase or thereafter, or at the start of the steady-state growth phase of the microorganism.

10. The method according to any one of claims 1 to 9, wherein whey is added to achieve a lactose concentration of at least 1% (by weight / volume).

11. The method according to any one of claims 1 to 10, wherein the whey is whey permeate.

12. The method according to any one of claims 1 to 11, wherein the whey is acidic whey, and the pH of the culture medium is adjusted to around pH = 7 during microbial culture.

13. The method according to any one of claims 1 to 12, wherein lactose accounts for at least 2% (by weight / by weight, wet weight) of the whey.

14. The method according to any one of claims 1 to 13, further comprising washing the microbial composition to remove the culture medium before heating in step iv) of the method for obtaining the casein composition.

15. The method according to any one of claims 1 to 14, further comprising dissolving the microbial composition before iv).

16. The method according to any one of claims 1 to 14, wherein the microorganisms in the microbial composition are not dissolved before step iv), and the heating in step iv) of the method for obtaining the casein composition dissolves the microorganisms.

17. The method according to any one of claims 1 to 16, further comprising 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 purified casein composition.

18. The method according to any one of claims 1 to 17, wherein the heating in step iv) of the method for obtaining a casein composition is performed at a temperature of 95°C or higher.

19. The method according to any one of claims 1 to 18, wherein the casein is selected from beta-casein, alpha-S1 casein, alpha-S2 casein, and mixtures thereof, and in particular the casein is selected from beta-casein, alpha-S1 casein, and mixtures thereof.

20. The method according to any one of claims 1 to 19, wherein the microorganism is transformed with a nucleic acid encoding alpha-casein and another nucleic acid encoding beta-casein, and the expression of the two nucleic acids is induced by lactose.

21. The method according to any one of claims 1 to 20, wherein the microorganism is not transformed with a nucleic acid encoding copper-casein.

22. The method according to any one of claims 1 to 21, wherein the casein accounts for at least 60%, preferably at least 62%, preferably at least 65%, preferably at least 67%, and preferably at least 70% of the total protein in the soluble fraction.

23. The method according to any one of claims 1 to 22, wherein the proportion of casein compared to the total protein is increased in the soluble fraction compared to the proportion in the microbial composition.

24. A method for culturing microorganisms, i. Preparing a microorganism transformed with at least one nucleic acid encoding casein, wherein the expression of the nucleic acid is induced by lactose, ii. The microorganism is cultured in a culture medium containing glucose as a carbon source, and the expression of the nucleic acid is not induced. iii. When the microorganism is in logarithmic growth, the glucose is depleted from the culture medium, and at the time of depletion, whey is present in the culture medium, thereby providing lactose present in the whey as a carbon source, and the expression of the nucleic acid is induced by the lactose present in the whey. iv. Further culturing the microorganism in the presence of a carbon source, Methods that include...

25. The method according to claim 24, wherein glucose is not present as a carbon source in iv.

26. The method according to claim 24, wherein whey is not present as a carbon source in iv.

27. The method according to claim 24, wherein in iv, both glucose and whey are carbon sources.

28. The method according to any one of claims 24 to 27, wherein the microorganism also metabolizes galactose as a carbon source.

29. The method according to any one of claims 24 to 28, wherein the nucleic acid encoding the casein is under the control of a promoter fused with the laco operator region of the lactose operon, and the microorganism expresses the lacI repressor of the operon.

30. The method according to any one of claims 24 to 29, wherein glucose and whey are present in the culture medium of ii.

31. The method according to any one of claims 24 to 29, wherein whey is not present in the culture medium of ii, and whey is added in iii.

32. The method according to any one of claims 24 to 31, wherein the depletion of glucose in iii is achieved by the consumption of glucose by the microorganisms without the resupply of glucose.

33. A method for reducing the environmental impact of a casein composition prepared by fermentation of transgenic microorganisms, i. Prepare a microorganism transformed with at least one nucleic acid encoding casein, ii. Culturing the microorganism using a medium containing whey so as to express and produce casein, iii. This provides a microbial composition in which the pH of the composition is 6.5 or higher, preferably less than 9, iv. Heating the microbial composition to reduce the amount of other proteins in the soluble fraction of the composition, wherein the heating is carried out at a temperature of 75°C or higher. v. The soluble fraction is recovered from the heated cell composition of iv) This allows obtaining a casein composition in the soluble fraction, and the environmental impact is reduced compared to the environmental impact of a casein composition obtained by the same process, except that glucose, sucrose, or molasses is used instead of whey in (b). Methods that include...

34. The method according to claim 33, wherein the reduction of environmental impact is observed in at least one criterion selected from (i) climate change, (ii) acidification, (iii) ecotoxicity, (iv) use of non-renewable energy resources, (v) eutrophication of freshwater, (vi) eutrophication of seawater, (vii) eutrophication of terrestrial areas, (viiii) toxicity to humans, carcinogenicity, (ix) toxicity to humans, non-carcinogenicity, (x) ionizing radiation, effects on human health, (xi) land use, (xi) use of material resources, (xiiii) ozone depletion potential, (xiv) formation of particulate matter, (xv) formation of photochemical oxidants, and (xvi) water use, preferably (i) climate change.

35. A method for obtaining a dairy substitute, comprising: performing the method according to any one of claims 1 to 23; mixing the casein composition with at least one other component comprising at least one component selected from the group consisting of protein, water, calcium, lipids, and carbohydrates to obtain a liquid precarious composition (LpCC); and further processing the liquid precarious composition (LpCC) to obtain a dairy substitute.

36. The LpCC process described above is vi. Adding at least one coagulant to the liquid composition in order to obtain a card, vii. Further processing the card to obtain a dairy substitute, wherein the dairy substitute is a cheese substitute or a yogurt substitute, The method according to claim 35, including the method described in claim 35.

37. The method according to claim 36, wherein the further step in vi) comprises acid precipitation of casein by heating at 90°C at room temperature or at pH = 4.

6.

38. A dairy product substitute that can be obtained by the method described in any one of claims 35 to 37.

39. The dairy substitute according to claim 38, wherein the aforementioned environmental impact is reduced in at least one of the criteria listed in claim 34.

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