Method for producing casein and use thereof

Abstract

The invention belongs to the food industry, relates to a novel method for producing casein compositions by culturing transgenic microorganisms in the presence of whey, and limits the environmental impact.

Description

Methods for producing casein and their uses Technical Field

[0001] This invention pertains to the food industry and relates to novel methods for producing casein compositions and their uses, particularly for producing dairy alternatives (especially vegan cheeses), while limiting environmental impact. Background Technology

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

[0003] However, dairy products are associated with several issues or concerns, including health, environmental, and ethical considerations. These concerns underscore the need for dairy alternatives to alleviate these diverse problems. Health issues include lactose intolerance, allergies, and other concerns such as high saturated fatty acid content, which is known to have potential negative health effects.

[0004] Furthermore, environmental and ethical issues associated with animal-derived food products have intensified over the past few decades. The burden of animal husbandry for a population of 7 billion (11 billion by 2050) is growing increasingly heavy. The environmental consequences are severe, primarily in terms of anthropogenic greenhouse gas (GHG) emissions, water consumption, wastewater pollution, and land occupation.

[0005] Today, cattle farming is considered one of the leading sources of GHG emissions, estimated to produce 7.1 gigatonnes of CO2 equivalent annually, accounting for 14.5% of all anthropogenic greenhouse gas (GHG) emissions.

[0006] The enormous water consumption is also related to animal agriculture. Furthermore, agricultural wastewater has a significant environmental impact. While the impact of crop fertilizers on groundwater cannot be ignored, the impact of animal agriculture wastewater is often enormous and has proven catastrophic in many parts of the world. In addition, livestock farming occupies nearly 80% of the world's agricultural land, but produces less than 20% of the global calorie supply, demonstrating the extremely high pressure that animal agriculture places on land resources.

[0007] Finally, animal welfare has become an increasingly important issue. The expansion of meat and dairy production and processing has transformed it into an intensive industrial process, which is increasingly seen as morally unacceptable.

[0008] Therefore, there is an urgent need for dairy alternatives to alleviate the above problems.

[0009] Plant-based alternatives are potential substitutes for traditional dairy products. However, these products are typically derived from starch, soy, almonds, or coconut milk and fall far short of mimicking the taste of dairy products. Furthermore, they differ fundamentally in their composition.

[0010] Therefore, there is a need today for dairy alternatives, and in particular cheese alternatives, that are: (i) free of unwanted compounds, (ii) similar in appearance, texture and taste to the original product, and (iii) nutritionally equivalent to or superior to the original product.

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

[0012] Table 1: Milk Composition Lipids and carbohydrates (except lactose) can be recovered from plants, while calcium can be recovered from inorganic, animal, or plant sources (such as seaweed). In the case of protein, other sources must be considered because the amino acid composition of animal protein differs from that of plant protein. Protein in milk can also be produced through fermentation, which is another source of non-animal-derived ingredients.

[0013] In addition, proteins isolated from milk are also used separately as nutritional supplements and in other applications. Therefore, milk proteins produced through fermentation, independent of other milk components, can also be used for applications beyond the manufacture of milk substitutes.

[0014] Fermentation-based production is based on the growth of microorganisms (bacteria or fungi) that produce compounds of interest in a fermenter, typically followed by the recovery and purification of those compounds. These microorganisms are often genetically modified, meaning they are transformed using gene constructs to express the desired protein or to obtain the compound through microbial metabolism. Protein production via fermentation is a method widely used in the food industry. Numerous studies have described the production of constituent proteins or homologs of milk via fermentation in various microorganisms (see below), and many have described the formulation of ingredients including fermented proteins to create dairy alternatives (US6270827; US5,942,274; WO2018039632; WO2020223700; WO2020081789, WO2020219596, WO2022098835).

[0015] Production via fermentation typically uses sugar feedstocks to support microbial growth and the production of desired compounds. Depending on the microorganisms' ability to metabolize different sugars, glucose, sucrose, or other sugars such as lactose can be used.

[0016] 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, and there is a certain preference level.

[0017] Many *E. coli* strains, including BL21, cannot consume sucrose. However, other strains, such as *E. coli* W (ATCC 9637), can efficiently utilize sucrose as a sugar source (Sabri et al., (2013). *Appl Environ Microbiol.* 79:478-87). In *E. coli*, two gene clusters are responsible for sucrose consumption. The *sc* regulator encodes a sucrose phosphotransferase system (Schmid et al., (1988). *Mol. Microbiol.* 2:1–8), while the *csc* regulator encodes a phosphotransferase-independent sucrose utilization system (Jahreis et al., (2002). *J. Bacteriol.* 184:5307-5316). *E. coli* strains that do not consume sucrose (such as BL21) can be converted into sucrose-consuming strains, for example, by introducing genes from the *csc* regulator (Bruschi et al., 2012. *Biotechnol Adv.* 20; 30(5):1001-10).

[0018] Microbial growth and metabolism require not only sugars but also nitrogen input. Various compounds can be added to this end, including liquid or gaseous ammonia, urea, amino acids, peptone, and yeast extracts.

[0019] Alternative raw materials derived from dairy products, such as whey, have also been used in the fermentation of various microorganisms, including Escherichia coli (González-Siso (1996) The biotechnological utilization of cheesewhey: A review. Bioresource technology Vol. 57, No. 1, pp. 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, Vol. 38, pp. 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. Vol. 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; Louasté 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 Polymorphism and 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, page 395; de Diviitis et al., (2023) Cheese-whey permeate improves the fitness.Biotechnology for Biofuels and Bioproducts, Volume 16:30). .

[0020] Whey is a byproduct or waste product of the cheese-making process, but it is also a byproduct or waste product of any other process, such as the production of Greek yogurt, some of which is drained from the product after curdling. In cheese production, 1 kg of cheese produces 10 liters of whey (Mollea et al., (2013) Chapter 24: Valorisation of cheese whey, a by-product from the dairy industry. pp. 549–588 in Food Industry. I. Mazzalupo, ed. InTechOpen, London, UK), and 1 kg of Greek yogurt produces about 2 to 3 kg of yogurt (Erickson, BE (2017) Chem. Eng. News, 95(6), 26-30).

[0021] The value utilization of whey, especially acid whey, is an important issue in the dairy industry (Gonzales-Siso (1996) The biotechnological utilization of cheese whey: a review. BioresourTechnol. 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. 104, 1262–1275). While some companies are able to realize the value of whey (especially soft whey, but sometimes also acid whey), acid whey is often discarded as waste.

[0022] Whey contains protein (whey protein, which makes up about 20% of the total protein in milk) and has a complex composition, including minerals and lactose. Whey is characterized by its yellow-green color due to the presence of riboflavin (vitamin B12; González-Siso (1996) The biotechnological utilization of cheese whey: Areview. Bioresource technology vol. 57, no. 1, pp. 1-11). In fact, whey contains a variety of 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 two main types of whey: sweet whey and sour whey.

[0023] Sweet whey is a byproduct associated with most cheese production, produced after enzymatic coagulation using natural or synthetic rennet. Its pH is approximately 6 to 7 (González-Siso (1996) The biotechnological utilization of cheese whey: A review. Bioresource technology, Vol. 57, No. 1, pp. 1-11), and it has a higher protein content and lower salt content compared to acid whey.

[0024] Acid whey is related to the fermentation of milk. Fermentation can be produced by the activity of lactic acid bacteria that lowers the pH by producing organic acids, or by 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 farm cheese and Greek yogurt, the production of which has increased significantly. Due to several inherent reasons, acid whey is difficult to monetize (Rocha-Mendoza et al., (2021) Acid whey trends and health benefits. J. Dairy Sci. Vol. 104, pp. 1262–1275), therefore, applying acid whey to fermentation has become a solution to this problem.

[0025] The whey in the composition can vary depending on the manufacturing process. For example, in acid whey, the lactose concentration can vary between about 2% and about 5% (by weight / volume) (Rocha-Mendoza et al., (2021) Acid whey trends and health benefits J. Dairy Sci. 104, 1262–1275; O'Donoghue and Murphy (2023) Comprehensive Reviews in Food Science and Food Safety, 22:2652-2677).

[0026] To realize the value of whey protein and lactose, various whey derivatives can be obtained.

[0027] Whey permeate was obtained after removing whey protein using ultrafiltration (O'Donoghue and Murphy (2023) op.cit.). Whey permeate can be used as a source of dairy solids in a variety of food, beverage, or feed applications.

[0028] In lactose-free whey permeate, most of the lactose is removed; however, lactose can still account for 60% of the dry weight, thus remaining an important part of the remaining components.

[0029] Whey can also be concentrated for easier transportation.

[0030] As long as the whey still contains at least about 2% lactose (w / w, wet weight), preferably at least 3%, preferably at least 4%, preferably at least 5%, preferably at least 8%, we will refer to such products as whey without distinction, whether they are undiluted (or whole) whey, permeate, or lactose-free permeate. In some embodiments, and when using concentrated whey, it may contain at least 30% lactose (w / w, wet weight), or at least 32% lactose, or 35% lactose or more.

[0031] Given the origin of whey, using such a raw material does not qualify for the production of animal-free products. However, using waste as a raw material can greatly contribute to achieving good economics and favorable life cycle analysis.

[0032] During fed-batch fermentation, the pH can be adjusted to maintain it within a specific range by adding 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).

[0033] All of these solutions are not exclusive and can be combined.

[0034] The inventors have proposed using whey as a carbon source for microbial cultures expressing casein, particularly during fed-batch cultures. They have demonstrated a reduction in environmental impact when using such whey products compared to the effects observed when using glucose, sucrose, or molasses as feedstock for microbial growth (and, where applicable, inducing casein production with isopropyl β-d-1-thiogalactoside (IPTG)). As mentioned above, given the composition of whey, the use of such products also provides the culture medium with vitamins and minerals, which is significant for sustaining long-term cell (especially bacterial) cultures.

[0035] It's important to note that fed-batch culture is a method of culturing microorganisms (such as bacteria, yeast, or cells) in a bioreactor to produce substances of interest. Compared to batch culture, fed-batch culture offers more robust control over the culture environment and can result in higher yields and product concentrations.

[0036] The process begins by inoculating a small number of microorganisms (inoculum) into a bioreactor containing a growth medium. This medium contains the nutrients (such as sugars, nitrogen sources, minerals, and vitamins) necessary for the growth of the microorganisms and the production of the desired product.

[0037] The bioreactor initially operates in batch mode, with all necessary nutrients added at the start of cultivation. During this period, microorganisms grow, consume nutrients, and also produce the target product. The culture medium does not contain excessive nutrients to avoid microbial overgrowth, the production of harmful byproducts, and to ensure better control.

[0038] As cultivation progresses, nutrient concentrations in the bioreactor decrease due to microbial consumption. During fed-batch processing, additional nutrients are gradually added to the bioreactor. The rate and amount of nutrient addition, along with various parameters such as temperature, pH, dissolved oxygen, agitation speed, and nutrient feed rate, are closely 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, thereby maintaining optimal growth conditions and extending the production phase. In continuous fed-batch processes, nutrients are continuously supplied. In other fed-batch processes, nutrients are supplied intermittently.

[0039] The culture is harvested once the desired product concentration is reached or after a given period of time. Harvesting should be carried out before the number of microorganisms decreases, as the death of such microorganisms may lead to the formation of undesirable products. The product can be purified and further processed.

[0040] The advantages of fed-batch processing include: controlling microbial growth and product formation by adjusting the nutrient feeding rate; achieving higher yields by providing nutrients in a controlled manner; extending production cycles; and reducing the formation of by-products. Summary of the Invention

[0041] In one embodiment, whey is present alone in the culture medium at the start of culture (inoculation period), or preferably together with another sugar (especially glucose, sucrose, fructose), particularly in the form of molasses.

[0042] In another embodiment, whey is not present in the culture medium at the start of culture (inoculation period). In this embodiment, another sugar is present in the culture medium, particularly glucose, sucrose, and fructose, especially in the form of molasses.

[0043] In one embodiment, whey is introduced as a carbon source during at least a partial feed batch. In one embodiment, no other sugars are provided with whey. In another embodiment, as described above, whey is introduced with another sugar.

[0044] In one embodiment, whey is present in the starting medium along with sugars other than lactose (especially glucose), and the fed-batch process includes providing additional nutrients, wherein for at least a period of time (ranging from 15 minutes to 1 hour, preferably from 20 minutes to 45 minutes, preferably about 30 minutes), sugars other than lactose (especially glucose) are provided again along with the additional nutrients, and only after this period of time are sugars other than lactose (especially glucose) provided again. In one embodiment, whey is not provided further along with sugars other than lactose (especially glucose). In another embodiment, whey is provided further along with sugars other than lactose (especially glucose).

[0045] In one embodiment, whey is not present in the starting culture medium, which contains only sugars other than lactose (especially glucose). In this embodiment, the fed-batch process includes providing additional nutrients, wherein whey is the only sugar provided along with the additional nutrients for at least a period of time (ranging from 15 minutes to 1 hour, preferably from 20 minutes to 45 minutes, preferably about 30 minutes). In this embodiment, after this period of time, sugars other than lactose (especially glucose) may be provided again along with additional nutrients. In one embodiment, whey is not provided further along with sugars other than lactose (especially glucose). In another embodiment, whey is provided further along with sugars other than lactose (especially glucose).

[0046] One goal is to use lactose, which is present in whey, as the sole sugar used by the microorganisms at certain points during cell culture. This also ensures that the microorganisms switch to casein production when casein expression is induced by lactose. Once casein production has begun, glucose or other sugars (even lactose as a sugar) can be reintroduced.

[0047] Given the lactose concentration in unconcentrated whey, an advantageous embodiment is to use a culture medium containing whey (up to 60%, preferably up to 55%, particularly about 45% to 55%, especially about 50% v / v) as the starting medium, which also contains sugars other than lactose, such as glucose, capable of inducing CCR (see below). The microorganisms will then use and consume another sugar. At a time specified by those skilled in the art (typically when the microorganisms are in the latter half of the exponential growth phase), no sugars other than lactose are supplied, thus allowing the microorganisms to consume the lactose present in the medium from the start of the culture (lactose was not consumed because the microorganisms used other sugars as a carbon source). This initiates lactose catabolism and deactivates CCR (thus allowing the consumption of lactose present in the medium and inducing casein production when it is lactose-inducible). Lactose is then consumed, and further carbon supply can be obtained by supplementing with any sugar (glucose, lactose, fructose, sucrose) without the need for further whey supply.

[0048] In some implementations, whey is used as the sole sugar source (lactose present in whey).

[0049] In other embodiments, whey is used in conjunction with other ingredients such as glucose (obtainable from corn or wheat) and sucrose (obtainable from sugar beets or sugarcane). These other ingredients can be pure sugar, molasses, or other forms.

[0050] It is preferable to use whey with other ingredients. In fact, because the concentration of lactose in whey is usually low, using such a product as the sole ingredient requires adding a large amount of whey to obtain a sufficient amount of lactose.

[0051] Furthermore, since whey contains lactose, when using a lactose-inducible promoter, lactose can trigger the expression of transgenes expressing 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 substance 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; deDiviitis et al., (2023) Cheese-whey permeate improves the fitness. Biotechnology for Biofuels and Bioproducts, Vol. 16:30).

[0052] In one implementation, it is recommended to use glucose and whey in the culture medium during the early stages of microbial growth (until the end of exponential growth or the beginning of stationary growth), with glucose preferentially utilized due to carbon catabolism inhibition (CCR). Once adequate microbial growth is achieved and recombinant casein expression is desired, the culture is switched to lactose catabolism (Görke and Stülke Nat Rev Microbiol 6, 613–624 (2008); Postma et al., Microbiol Rev. 1993 Sep;57(3):543-94). Low levels of glucose can be added to the culture to maintain adequate carbon supply for viable microbial growth without switching back to glucose-only to continue protein expression. Although other sugars (such as sucrose or galactose) can be used, glucose is preferred, at least at the beginning of the culture (exponential growth phase) to induce CCR, especially when a lactose-inducible promoter is used to trigger casein expression.

[0053] Lactose is a disaccharide composed of glucose and galactose. Lactose metabolism typically depends on the hydrolysis of both glucose and galactose. Some *E. coli* strains, such as BL231(DE3), cannot consume the galactose portion because mutations result in a lack of the enzyme involved in galactose metabolism via the Leloir pathway. Therefore, to utilize lactose entirely as a bacterial carbon source at maximum yield, strains with a complete Leloir pathway can be used, and preferably used (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 the pathway can be restored in mutant strains through genetic engineering. Such strains capable of metabolizing D-galactose to form glucose are typically engineered as gal+ strains.

[0054] Furthermore, the inventors used suitable purification methods to produce casein of a grade compatible with food industry use, which has high purity and can be easily scaled up at the industrial level at a cost matching the development, particularly in terms of energy consumption.

[0055] Specifically, recombinant casein can be isolated from biomass and culture medium by heating the composition, as disclosed in WO2022253816.

[0056] Compared to the effects observed when using glucose, sucrose, or molasses as the carbon source for culturing microorganisms (i.e., whey, especially acidified whey or acidified whey permeate, is not used in this method), and particularly compared to the effects of using glucose, the method disclosed herein results in a casein composition with reduced environmental impact. Furthermore, this provides the dairy industry with a way to recycle waste that currently requires treatment and disposal. When using microorganisms for lactose-induced casein expression, the time point at which lactose in whey induces casein expression is determined by IPTG induction of protein expression.

[0057] Environmental impacts are multifactorial and must be considered from aspects such as climate change, freshwater pollution, seawater pollution, soil and air pollution, impacts on ecosystems, fossil resource use, material resource depletion, and water use. Standards may include: (i) climate change, measured in kg CO2 equivalents (eq represents equivalent), and (ii) acidification, measured in mol H₂. + -eq, (iii) ecotoxicity, in comparative ecosystem toxicity units (CTUe), (iv) utilization of non-renewable energy sources (in MJ or kWh), (v) freshwater eutrophication (in kg P-eq) (kg phosphorus equivalent), (vi) seawater eutrophication (in kg N-eq) (kg nitrogen equivalent).

[0058] (vii) Terrestrial eutrophication (cumulative excess in mol N-eq), (viii) Human toxicity, carcinogenicity (in human comparative toxicity units, CTUh), (ix) Human toxicity, non-carcinogenicity (in human comparative toxicity units, CTUh), (x) Ionizing radiation, effects on human health (compared to U 235 Compared to human exposure efficiency, in kNq U 235 -eq), (xi) Land use soil quality index (ss), (xii) Material resource use (kg Sb-Eq), (xiii) Ozone depletion potential (kg CFC11-eq), (xiv) Particulate matter formation (disease incidence), (xv) Photochemical oxidant formation (kg NMVOC-eq), (xvi) Water use (depletion world equivalent m 3 ).

[0059] The significance of this method lies in providing improvements for at least one of the aforementioned criteria, particularly climate change, acidification, eutrophication (v, vi, and / or vii), and / or water use. Notably, improvements can be achieved for the impacts of climate change as well as for at least another criterion.

[0060] Therefore, the present invention also relates to a method for reducing the environmental impact of preparing a casein composition by fermentation of transgenic microorganisms, comprising: a. providing microorganisms transformed with at least one nucleic acid encoding casein; b. culturing said microorganisms in a whey-containing medium to express and produce casein; c. thereby providing a microbial composition wherein the pH of said composition is equal to or higher than 6.5, and preferably lower than 9; d. heating said microbial composition to reduce the amount of other proteins in the soluble fraction of said composition, wherein heating is carried out at a temperature equal to or higher than 75°C; e. recovering the soluble fraction from the heated cell composition of iv), thereby obtaining a casein composition from said soluble fraction, wherein the environmental impact is reduced compared to that of a casein composition obtained by the same steps except that glucose or sucrose or molasses is used instead of whey in step b.

[0061] Specifically, a reduction in environmental impact is observed in at least one of the following criteria: (i) climate change, (ii) acidification, (iii) ecotoxicity, (iv) use of non-renewable energy resources, (v) freshwater eutrophication, (vi) seawater eutrophication, (vii) terrestrial eutrophication, (viii) human toxicity, carcinogenicity, (ix) human toxicity, non-carcinogenicity, (x) ionizing radiation, impact on human health, (xi) land use, (xii) material resource use, (xiii) ozone depletion potential, (xiv) particulate matter formation, (xv) photochemical oxidant formation, and (xvi) water use, preferably (i) climate change.

[0062] Casein compositions can also be processed to obtain dairy alternatives, such as cheese and yogurt derived from curd, as well as ice cream and reconstituted milk; the environmental impact of these dairy alternatives is also reduced compared to the environmental impact observed when using recombinant casein obtained from microorganisms grown in the presence of glucose, sucrose, or molasses as carbon sources. This method generally exhibits better environmental impact across almost all standards compared to the same dairy alternatives made using recombinant casein obtained from microorganisms grown in the presence of glucose as the sole carbohydrate source. The environmental impact is also improved across many standards when using recombinant casein obtained from microorganisms grown in the presence of sucrose, and even when using recombinant casein obtained from microorganisms grown in the presence of molasses.

[0063] In the context of this invention, "casein" refers to any casein or mixture of caseins. Therefore, "casein" means α-S1 casein, α-S2 casein, β-casein, or κ-casein. To some extent, it can also refer to any mixture of these proteins. The terms "casein" or "caseins" are generally used to discuss casein proteins.

[0064] In the context of this invention, the term "between" includes end values.

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

[0066] In the context of 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 that contains more than 80% water (the ratio of water to the total mass of the fat-free product) on a fat-free basis and between 2% and 15% of the total weight (the ratio of protein mass to the total mass of the product).

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

[0069] In the context of this invention, the term "liquid pre-curled milk composition" or "LpCC" refers to a composition comprising at least one casein and at least one other ingredient prior to the addition of rennet and fermentation products and to curdling (particularly casein coagulation), said other ingredient comprising at least one of water, calcium, lipids, and carbohydrates. The casein concentration in LpCC is higher than that in milk. The concentrations of other ingredients in LpCC are also higher than those in milk. This allows for water savings when using LpCC with recombinant casein as disclosed in this application, since LpCC does not represent artificial (cow's) milk. The concentrations of the protein, lipids, calcium salts, and / or carbohydrates are adjusted to suit the final desired composition of the cheese substitute.

[0070] In the context of this invention, the term "coagulant" refers to a chemical or biochemical composition capable of triggering coagulation. Coagulation can be achieved by adding an acid solution, by adding a starting culture (ferments that grow in the presence of carbohydrates and cause a decrease in pH), by heat treatment, by adding a calcium chelating agent, by adding a natural or recombinant rennet, by adding a rennet substitute (e.g., animal protease or plant rennet), or by a combination of these processes. Coagulants can be created by adding any additive, compound, composition, or treatment agent that induces coagulation, which can be used alone or in combination with another additive, compound, composition, or treatment agent. When preparing an edible composition that does not contain animals, it is preferable to use an acidifier (such as an acid or a starting culture) to avoid the use of rennet, so that the coagulant does not contain any animal-derived elements. It is advantageous when the acidifier contains one or more lactic acid bacteria, but acidic chemicals can also be used.

[0071] In the context of this invention, the term "curd" means a composition in which casein coagulates or precipitates under the action of a curdling agent, and can be separated from the liquid phase (if present) by draining (e.g., on a cheesecloth). Curd also contains other ingredients, including water and lipids (if present).

[0072] In the context of this invention, the term "fermenter" refers to a composition containing at least one strain of microorganism added in a process for producing a cheese substitute. Lactic acid fermenters are responsible for lactic acid fermentation, which significantly leads to acidification of the culture medium. Therefore, lactic acid fermenters can be used as curdling agents.

[0073] Other fermented ingredients are used in cheese production to process the curd, thereby altering its texture, flavor, aroma, and chemical composition (especially by breaking down proteins into smaller peptides). These fermented ingredients, which may be called "ferments for maturation," "maturation ferments," or "ripening ferments," are generally not used in the production of fresh cheese. These maturation ferments can be added along with the curdling agent.

[0074] In the context of this invention, the term "non-animal-derived" means a compound or composition that is not directly derived from an animal, is not produced from cultured animal cells, or is not isolated from animal products (such as milk). Thus, compounds or compositions produced by microbial fermentation are "non-animal-derived," even if some animal-derived products, such as bacterial peptones, may be involved in the fermentation process. Therefore, in the context of this invention, when a protein naturally occurring in an animal is produced in microbial (such as bacterial or yeast) cells or plant cells, that protein will be referred to as non-animal-derived, even if its sequence or structure may be identical to that of proteins isolated from animals.

[0075] In the context of this invention, the term "animal-free" means a compound or composition that is not derived from animals, cultured animal cells, or animal products (such as milk), and whose production method does not involve any animal-derived raw materials or additives.

[0076] In the context of this invention, the term "texturing agent" means any gelling agent, including emulsifiers such as lecithin, and hydrocolloids such as cinnamon gum, guar gum, tamarind gum, guar gum, fenugreek gum, gum arabic, agar (agar agar or agar-agar), carrageenan, astragalus gum, xanthan gum, carob (sophora bean) gum, and cellulose gum.

[0077] In a first aspect, this document discloses a method for obtaining a casein composition, comprising: i. providing microorganisms transformed with at least one nucleic acid encoding casein; ii. culturing said microorganisms in a whey-containing medium to express and produce casein; iii. thereby providing a microbial composition wherein the pH of said composition is equal to or greater than 6.5, and preferably less than 9; iv. heating said microbial composition to reduce the amount of other proteins in the soluble fraction of said composition, wherein the heating is performed at a temperature equal to or greater than 75°C; and v. recovering the soluble fraction from the heated cell composition of iv), thereby obtaining a casein composition in said soluble fraction.

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

[0079] The method enables a reduction in environmental impact for at least one of the aforementioned criteria (particularly greenhouse gas (GHG) emissions, in CO2 equivalent mass) compared to methods performed in (ii) using glucose, sucrose, or molasses instead of whey. This impact can be particularly reduced when acidic whey (or its derivatives, such as permeate) is used as whey in step (ii).

[0080] Specifically, compared to using glucose, it is possible to achieve a reduction of at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, or possibly at least 30%, depending on the criteria examined.

[0081] Reduced environmental impacts can also be observed in dairy alternatives made using casein obtained through the methods disclosed herein.

[0082] It's important to note that microorganisms, especially bacteria, exhibit an exponential (or logarithmic) phase, during which cells multiply after each generation. Metabolic activity is high during this phase, and the energy (nutrients) in the culture medium direct this multiplicative activity. After the exponential phase, the population growth experienced in the logarithmic phase begins to decline, and cell growth reaches a plateau or stationary phase, in which the number of dividing cells equals the number of dying cells.

[0083] In the context of the process disclosed herein, whey is preferably used as the feedstock for the microorganisms, preferably at the mid or late exponential phase or at the beginning of the stationary phase (at which point the supply of sugars other than lactose is avoided and whey is supplied instead, or whey is present in the culture medium from the inoculation period), and sometimes this supply is maintained during casein production. However, an early induction step is also conceivable. Those skilled in the art can determine the end of the exponential phase or the beginning of the stationary phase for microbial culture, depending on the doubling time of the microorganisms, the concentration of the starting culture, and the duration of growth. Measuring the optical density (OD) of the culture can also provide information about the microbial growth phase. When using *E. coli* as the microorganism, the exponential phase is conceivable to last between 16 and 20 hours, followed by a stationary phase of approximately 7 to 10 hours for protein expression.

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

[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 culture 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 culture medium using molasses or distiller's grains as a carbon source. In one embodiment, fermentation is carried out in a culture 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 culture medium as a nitrogen source. In one embodiment, fermentation is carried out on a culture medium containing ammonia, urea, amino acids, peptone, or yeast extract as a nitrogen source. In one embodiment, fermentation is carried out on a culture medium containing whey as the primary raw material (primary carbon source). In one embodiment, fermentation is carried out on a culture medium containing acidified whey or acidified whey permeate. In one embodiment, fermentation is carried out on a culture medium containing whey and another sugar as an additional sugar source. In a preferred embodiment, fermentation is carried out on a culture medium containing acidified whey (or acidified whey permeate) and glucose, sucrose, or lactose as additional sugar sources. Glucose can be used as another carbon source.

[0086] Before heating, or during microbial growth, the pH can be adjusted by adding liquid or gaseous ammonia, sodium hydroxide, potassium hydroxide, calcium hydroxide, or other alkaline compounds. The pH is above 6.5, and preferably below 9.

[0087] The microorganism is genetically modified and contains at least one transgene (nucleic acid) encoding a casein, which is introduced into and present in the microorganism. This transgene can be present within the microbial genome or outside the genome (on plasmids, granules, artificial chromosomes, such as bacterial artificial chromosomes or yeast artificial chromosomes).

[0088] In one specific embodiment, the microorganism is a bacterial cell, particularly *Escherichia coli*. In another embodiment, the microorganism is a fungal cell (including yeast cells). In yet another embodiment, the microorganism is a eukaryotic cell, particularly a plant cell. Specifically, suitable prokaryotic hosts for casein expression 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 can also be used for protein production.

[0089] Preferably, casein is produced in non-animal cells, and more preferably in bacterial cells.

[0090] Microorganisms transform their cells with at least one nucleic acid used to express casein. This nucleic acid contains elements necessary for the transcription and translation of the transgene encoding casein. Specifically, the nucleic acid should contain a promoter sequence, a sequence encoding casein, and a terminator sequence.

[0091] In some implementations, the microorganism transforms (and produces) a casein with a nucleic acid encoding a casein. It is preferred when the expression of the nucleic acid (protein production) is lactose-induced, i.e., the expression of the casein-encoding nucleic acid in the presence of lactose metabolized by the microorganism (lactose-induced expression).

[0092] In other embodiments, the microorganism expresses two casein proteins. In such embodiments, the microorganism can be transformed with two transgenes, each for the expression of a given casein protein. It is preferred when the expression of at least one transgene is lactose-induced. Preferably, both transgenes are expressed lactose-induced (using the same or different systems). In another embodiment, the two transgenes are linked together in an operon (thus producing two casein proteins). It is preferred when the expression in the operon is lactose-induced.

[0093] An implementation scheme for microbial expression of more than two casein proteins has also been envisioned. In this implementation scheme, each transgene encoding a given casein can be expressed under the control of its own promoter (preferably for lactose-induced expression), or some or all of the transgenes can be co-expressed in the operon (preferably for lactose-induced expression).

[0094] In some implementations, the microbial culture contains a mixture of microorganisms transformed with a nucleic acid encoding a casein and other microorganisms transformed with a nucleic acid encoding a different casein.

[0095] Lactose-induced gene expression is known in the art. It is typically based on the lactose operon (lac operon), which contains an operon region (laco). In the absence of lactose, the lac repressor protein lacI binds to the operon region, thereby preventing transcription of genes downstream of the laco region. In the presence of lactose (isolactose, an isoform of lactose), the lacI protein undergoes a conformational change and no longer binds to laco, thus allowing transcription of the downstream sequence of the operon. The sequence of the lac operon is TTGTGAGCGGATAACAA (SEQ ID NO: 5). This system (containing the laco operon and the lacI repressor protein) can be used for the expression of genes downstream of the laco operon, where the promoter upstream of the laco operon can be the natural promoter of the lac operon or another promoter (such as the T7 promoter). The lac operon contains regulatory regions including a promoter (P), to which RNA polymerase binds and initiates transcription; an operon (O), to which the lac repressor protein binds and prevents transcription of the structural gene in the absence of lactose; and the lacI gene, which encodes the lac repressor protein, which binds to the operon and blocks transcription in the absence of lactose.

[0096] It can be noted that lactose-inducible promoters and systems have been described in the prior art. The lac promoter, which regulates the expression of the lactose operon (lac operon), can be cited, as it is essential for lactose transport and metabolism in *E. coli* and many other enteric bacteria. The P(bgaL) promoter (Hartman et al., *Appl Environ Microbiol.* 2011 Jan;77(2):471-8), and the P promoter disclosed in Zhang et al., (*Biochemical Engineering Journal* Volume 151, November 15, 2019, 107316), can also be cited. lacA and P lacLM The promoter, or the P promoter disclosed by Heiis et al. (Microb Cell Fact 15, 50 (2016)). lacA Promoter.

[0097] In some implementations, the microorganisms are able to use galactose as a carbon source. Specifically, it is preferred when the microorganism expresses the galETKM operon from the *Escherichia coli* K12 strain, which is essential for galactose consumption. Notably, the galETKM operon contains four genes: galE encodes an epimerase, galT encodes uridyltransferase, galK encodes galactokinase, and galM promotes the interconversion between β-galactose and α-galactose.

[0098] Therefore, in some embodiments, bacteria are cultured where casein is controlled by a lactose-inducible promoter (e.g., a promoter fused to the laco region and the bacteria express the lacI repressor protein), and the bacteria also possess the genetic mechanisms required for galactose consumption. In this embodiment, the bacteria are thus able to efficiently consume glucose, lactose (after CCR), and galactose produced from lactose consumption.

[0099] In some embodiments, a method for culturing microorganisms may be cited, comprising: providing microorganisms transformed with at least one nucleic acid encoding casein, wherein the nucleic acid is cultured under the control of a lactose-inducible promoter in the presence of glucose and optionally in the presence of whey, preferably until the exponential growth phase, reducing the amount of glucose, particularly when the microorganisms are in the exponential growth phase, culturing the microorganisms using a whey-containing medium to induce the expression of a gene encoding casein and the production of casein (glucose depletion will relieve the catabolism inhibition of the lactose-inducible promoter), optionally further culturing the microorganisms in the presence of glucose after inducing casein expression and production, and optionally recovering the composition of the cultured microorganisms that have expressed and produced casein. This method includes the various steps described elsewhere herein. It enables the induction of casein expression when the cells are suitable for such expression (primarily during the exponential growth phase, approximately in the middle of this phase) and provides the cells with an optimal carbon source while reducing cost and environmental impact. This yields a culture of a casein-expressing microorganism (preferably bacteria).

[0100] Specifically, a method for culturing microorganisms can be performed, comprising: i) providing microorganisms transformed with at least one nucleic acid encoding casein, wherein expression of the nucleic acid is induced by lactose; ii) culturing the microorganisms in a medium containing glucose as a carbon source, wherein expression of the nucleic acid is not induced; iii) depleting the glucose in the medium while the microorganisms are in the exponential growth phase, wherein whey is present in the medium at the time of depletion, thereby providing lactose present in whey as a carbon source, and wherein expression of the nucleic acid is induced by lactose present in whey; and iv) further culturing the microorganisms in the presence of the carbon source.

[0101] In one implementation, glucose is not present as a carbon source in IV.

[0102] In one implementation, whey is not present as a carbon source in IV.

[0103] In one implementation, the carbon source in iv comprises glucose and whey.

[0104] In one implementation, as disclosed elsewhere, the microorganisms also metabolize 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 operon region of the lactose operon, and the microorganism expresses the lacI repressor protein of said operon. This ensures inhibition of the expressed catabolism in ii. Therefore, particularly in this embodiment, glucose and whey may be present in the culture medium of ii.

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

[0107] In one implementation, glucose depletion in iii is achieved by the microorganism consuming glucose without glucose replenishment (while a continuous supply of glucose or other carbon sources is typically required during culture; stopping this replenishment will result in the complete depletion of previously supplied glucose, and the microorganism will then begin using whey as a carbon source).

[0108] In one implementation, the microorganism is bacteria, particularly Escherichia coli.

[0109] When the method includes the following steps, it may obtain a casein composition: thereby providing a microbial composition thus obtained, wherein the pH of the composition is equal to or higher than 6.5, and preferably lower than 9; 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 equal to or higher than 75°C; and recovering the soluble fraction from the heated cell composition, thereby obtaining a casein composition in the soluble fraction.

[0110] As described above, specific embodiments include cases where a starting composition (a composition containing casein and other proteins) has already been obtained from a bacterial culture. In such embodiments, as described above, the bacteria have been transformed with one or more nucleic acids encoding one or more caseins.

[0111] In particular, in one implementation, the bacteria have been transformed with one or more nucleic acids encoding β-casein.

[0112] In particular, in one implementation, the bacteria have been transformed with one or more nucleic acids encoding α-S1 casein.

[0113] In particular, in one implementation, the bacteria have been transformed with one or more nucleic acids encoding α-S2 casein.

[0114] In particular, in one embodiment, the bacteria have been transformed with one or more nucleic acids encoding β-casein and α-S1 casein.

[0115] In particular, in one embodiment, the bacteria have been transformed with one or more nucleic acids that encode β-casein, α-S1 casein, and α-S2 casein, or a combination of two of them.

[0116] It should be noted that when producing multiple proteins, bacteria can be transformed with different nucleic acids (each encoding a different protein) or with a single nucleic acid (containing elements that allow the production of various proteins, such as operons). Such methods of transforming microorganisms and bacteria specifically for the production of one or more proteins are known in the art.

[0117] Therefore, the recovered casein composition contains casein, preferably selected from β-casein, α-S1 casein, α-S2 casein, and mixtures thereof, especially from β-casein, α-S1 casein, and mixtures thereof. As previously described, the microorganism is transformed with a nucleic acid encoding α-casein and another nucleic acid encoding β-casein, wherein both genes are under the control of a lactose-inducible promoter.

[0118] It is preferred when the microorganisms are not transformed with nucleic acids encoding κ-casein. Therefore, it is preferred when the microorganisms do not produce κ-casein and when κ-casein is not present in the various compositions and solutions.

[0119] In the method disclosed in this paper, whey serves as an important source of carbon required for microbial growth.

[0120] In one implementation, microbial culture is initiated in the presence of sugars other than lactose, as described above, particularly in the presence of glucose. When bacteria, especially *E. coli*, are used as the microbe, glucose is present in an amount sufficient to induce CCR (carbon catabolism repression). Indeed, the presence of both glucose and lactose leads to a regulatory scenario in which the lac operon is repressed. Glucose is highly preferentially utilized by catabolism repression, keeping cAMP levels low and essentially reducing or eliminating transcriptional activity of the lac operon.

[0121] In this situation, bacteria use glucose as a carbon source, while lactose metabolism is inhibited. It is important to note that lactose metabolism involves the breakdown of one molecule of lactose into one molecule of glucose and one molecule of galactose. When glucose is present as a carbon source in the culture medium, this metabolism is inhibited because the catabolites produced by glucose breakdown prevent the activation of the enzymes required for lactose metabolism. However, once these enzymes are activated, inhibition no longer occurs (Görke and Stülke Nat RevMicrobiol 6, 613–624 (2008); Postma et al., Microbiol Rev. 1993 Sep;57(3):543-94).

[0122] Specifically, it is preferable to use glucose (or a sugar other than lactose) at the beginning of the culture and during the exponential growth phase, as this will ensure that the bacteria grow at an appropriate exponential rate.

[0123] Whey should be used as a lactose source to induce casein expression (if a lactose-inducible system is used) or as a raw material for stable protein growth during the later stages of exponential growth or at the beginning of steady growth.

[0124] To trigger the switch to lactose metabolism and consumption (stopping CCR), the amount of glucose must be restricted (even to the point of depleting glucose from the culture medium). Once this switch has occurred, glucose or another sugar, such as sucrose, fructose, or galactose, can be added back to the culture medium, especially in the form of molasses, to replenish the sugar supply.

[0125] In one implementation, whey is absent from the culture medium at the start of cultivation, and whey is added when the amount of glucose is limited and a transition to lactose consumption (and induction of protein expression) is desired (during the exponential phase or at the start of the stationary phase).

[0126] In another implementation, whey is added along with glucose at the start of culture and at the beginning of the exponential phase. Because the glucose concentration is maintained at a high level sufficient to induce CCR, the whey is not consumed. Therefore, the switch to lactose consumption is achieved by reducing glucose feeding for a period of time (30 minutes may be sufficient), and then the bacteria utilize the lactose present in the whey. A small amount of glucose can then be added again to prevent glucose accumulation in the culture medium (this can be verified by sampling the medium and measuring the glucose concentration). Typically, stopping glucose feeding to inhibit CCR, followed by refeeding with glucose, does not cause CCR to reappear.

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

[0128] Other sugars besides glucose can also be used to initiate culture, such as glycerol, 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), with whey added later in the culture at an appropriate time (especially to induce casein expression). The choice of sugar and the specific method of adding whey can be readily designed by those skilled in the art.

[0129] In one embodiment, microbial culture is carried out in a fed-batch reactor, with feeding provided at periodic intervals. In another embodiment, the culture is carried out in a continuous mode, accompanied by continuous feeding and product removal.

[0130] In another implementation, cultivation is carried out in batches.

[0131] It is important to note that during fed-batch fermentation, substrates and supplements are added to the fermentation medium in a controlled manner, which is the opposite of batch fermentation, in which all substrates are present in the bioreactor at the start of the process.

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

[0133] When lactose consumption occurs, the lactose concentration in the culture medium is preferably higher than 1%, and more preferably between 1% and 5% (w / v, corresponding to 1 g / 100 mL to 5 g / 100 mL).

[0134] In one implementation, whey is undiluted whey or whole whey, i.e., the liquid obtained from milk curdling or filtration. Whey contains some whey protein.

[0135] In another embodiment, whey is permeated whey obtained by ultrafiltration of whole whey, in which serum proteins of whey are removed.

[0136] In one embodiment, acid whey (or osmotic solution derived therefrom) obtained by acidifying coagulated milk with lactic acid bacteria is used. In this embodiment, alkali is added to the culture medium to maintain the pH of the solution containing microorganisms at approximately pH 7.

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

[0138] In one embodiment, a composition containing casein and other proteins (and possibly microorganisms obtained through microbial culture) flows from a first container through a device containing a pipe or tube, such a tubular exchanger having the dual function of transferring the composition and heating it. Thus, heat transfer (or heat exchange) occurs during the composition's passage through the pipe (corresponding to heating step iv) to heat the composition; this transfer takes place at least partially on the pipe. This heating precipitates the other proteins present in the solution, while the casein remains in the soluble fraction.

[0139] Soluble fractions can be recovered by centrifuging the liquid composition flowing from the pipeline.

[0140] Compared to the starting composition, the concentration of casein and / or the ratio of casein to other proteins is higher in the recovered soluble fraction, thereby enriching casein in the soluble recovered fraction.

[0141] As indicated, the composition of i) contains soluble and insoluble fractions. The soluble fraction refers to the fraction that does not precipitate upon centrifugation. The insoluble fraction is the precipitate obtained after centrifugation. Centrifugation can be carried out at approximately 3000 g for 20 to 30 minutes. On an industrial scale, the soluble fraction can be recovered using continuous flow centrifugation or other methods such as filtration. In the case of filtration, different types of membranes can be used.

[0142] Preferably, the desired technical effect is achieved by heating during passage through the pipe in the absence of organic solvents or with the addition of organic solvents.

[0143] The device may include a zone (such as a chamber or a pipe of a certain length) preceding the activated carbon zone for preheating the composition so that it subsequently flows into the pipe at a desired temperature. For example, preheating can raise the temperature of the composition to approximately 80°C when it is at room temperature. The device may also include a zone after the heated pipe zone for cooling the composition at a temperature below 100°C (preferably up to approximately 60-80°C) to enable the recovery of soluble fractions.

[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] The other proteins are non-casein proteins. Therefore, heating makes it possible to obtain a casein-rich composition. Casein enrichment in a composition refers to the increase in casein in the soluble fraction compared to the amount of casein present in the soluble fraction of the initial composition before it flows through the pipe.

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

[0147] For example, microbial cells (preferably bacteria) can be centrifuged after cultivation to wash away the precipitate, and then resuspended in a suitable liquid or fluid (a suitable buffer, more preferably water, and more preferably free of organic solvents). The pH of the buffer is not acidic (equal to or higher than 6.5), preferably close to neutral (between 6.5 and 7.5), or alkaline (preferably lower than 9). In fact, casein precipitates in acidic pH environments. It is also preferred that the buffer is nonionic or has low ionic strength.

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

[0149] In one embodiment, the microorganisms in the microbial composition have been lysed prior to iv to obtain a liquid composition, wherein the liquid composition is in a first container. In another embodiment, the microorganisms in the microbial composition are not lysed and are lysed by heating in iv).

[0150] Microbial lysis can be performed using any method known in the art, preferably chemical lysis (such as detergents), enzymatic lysis (e.g., using lysozyme or proteinase K), or mechanical methods (using a high-pressure homogenizer).

[0151] Optionally, a further step may be performed, selected from adding activated carbon, chemical resin, membrane filtration (ultrafiltration, nanofiltration, or reverse osmosis), centrifugation, chromatography, or precipitation of casein to the soluble fraction recovered from v). ​​In particular, the soluble fraction can be recovered by centrifuging the liquid composition exiting the pipeline using a conventional or disc centrifuge. Alternatively, the soluble fraction may be obtained from microfiltration or diafiltration.

[0152] Activated carbon can be added to the soluble fraction and stirred. The activated carbon can be used to adsorb impurities (especially organic impurities or chlorine) present in the soluble fraction that were not removed during the recovery of the soluble fraction in iii). It is preferable to stir when adding the activated carbon and to remove the activated carbon before proceeding with other methods (e.g., those disclosed below) on the soluble fraction.

[0153] Chromatography is widely used in purification methods, especially affinity chromatography, ion exchange chromatography, and hydrophobic interaction chromatography.

[0154] Membrane filtration, primarily including ultrafiltration and nanofiltration, is also widely 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, Vol. 145, pp. 1-22), and membrane filtration technology is widely used in the dairy industry. Interestingly, they can also be used to separate different casein proteins from each other (see above).

[0155] Finally, further purification can be achieved by taking advantage of the specific properties of caseins (Post et al. (2012) Effect of temperature and pH on the solubility of caseins: Environmental influences on the dissociation of caseins. J. Dairy Sci. Vol. 95: 1603–1616), especially their tendency to precipitate under acidic conditions.

[0156] In a preferred embodiment, the soluble fraction is further processed by precipitating casein under acidic conditions. After precipitation, the casein can be resuspended and can also be redissolved using a suitable alkaline 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. Vol. 95: 1603–1616). In a preferred embodiment, the soluble fraction is further processed by precipitating casein in a pH range of pH 4 to pH 5. In a more preferred embodiment, casein precipitation is carried out at approximately pH 4.6. Various acids can be used, including lactic acid, hydrochloric acid, and sulfuric acid.

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

[0158] When isolating casein from microbial cultures, precipitation under acidic conditions is particularly beneficial because the acidic conditions allow for the removal or degradation of any nucleic acids that may be present in the casein composition. The presence of microbial DNA or RNA has proven potentially harmful (at least from a regulatory perspective) when casein is used to obtain edible compositions intended for human consumption (e.g., cheese substitutes). pH values ​​should be in the range of approximately 4.6, temperatures from 80°C to 140°C, and time intervals from a few minutes to 2 hours.

[0159] Alternatively, nucleases, such as DNases, can be used to eliminate recombinant DNA; nucleases can be removed by heating.

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

[0161] Subsequently, the pH can be neutralized to obtain casein salts. For this purpose, various basic compounds can be added, such as liquid or gaseous ammonia, sodium hydroxide, potassium hydroxide, or calcium hydroxide. The target pH can be between 6.5 and 9. The pH adjustment kinetics can be continuous or after a series of plateau periods to achieve a stable target pH. Prior to 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 one preferred embodiment, sodium hydroxide is used to adjust the pH. In another embodiment, potassium hydroxide is used to adjust the pH. In yet another embodiment, calcium hydroxide is used to adjust the pH.

[0163] In one specific embodiment, the casein composition is dried. When obtained from bacterial cultures, this composition should contain approximately 15% to 30% casein (w / w) and carbohydrates in the dried composition.

[0164] Preferably, a small amount of water is retained to facilitate subsequent rehydration. Therefore, desiccation should be understood as reducing the amount of water. In some embodiments, the amount of water is about 50% or less (w / w).

[0165] This insoluble fraction contains most of the biomass produced during fermentation and can be processed and multiplied separately. Before being used in feed or food applications, this biomass can be treated using the same methods as casein treatment to remove recombinant DNA: pH range 4.6, temperature from 80°C to 140°C, and time from a few minutes to 2 hours; alternatively, nucleases, such as DNases, can be used to eliminate the recombinant DNA, and these nucleases can be removed by heating.

[0166] Therefore, in the disclosed method described above, the combined stream of i) is heated at a temperature above 75°C (heating temperature) as it flows through the pipe. More preferably, this temperature is above about 80°C, more preferably above about 85°C, more preferably above about 90°C, more preferably above about 95°C, and most preferably above about 100°C, or above 100°C. Temperatures equal to or above 105°C, or equal to or above 110°C, may be suggested. Temperatures up to 140°C or even 150°C may be used. Preferably, the composition is heated for at most 3 minutes, or even at most 1 minute. In this case, a suitable temperature is preferably between 95°C and 115°C, preferably between 100°C and 115°C (including the endpoints). A temperature of about 110°C would be suitable. Such temperatures enable the technical and functional effects of removing other proteins, thereby reducing the amount of other proteins and / or transferring casein from the insoluble fraction to the soluble fraction, while the heating duration is short (at most five minutes, preferably at most two minutes, but can be 90 seconds or less, or 75 seconds or less, more preferably 60 seconds or less, or even 45 seconds or less). Examples show that durations of 30 seconds or even 2 seconds can be applied. However, it is preferred to heat the composition flowing in the pipe for at least 5 seconds or at least 10 seconds.

[0167] The pressure within the conduit is typically higher than 3 bar, more preferably 4 bar or higher, and even more preferably 4.5 bar or higher. It is typically 7 bar or lower, preferably 6.5 bar or lower, and even more preferably 6 bar or lower. A pressure between 4.5 bar and 5.5 bar (including the endpoints) is suitable.

[0168] When referring to measurable values, the term “about” means to cover a variation of ±3% from the specified value, as such variation is appropriate for implementing the disclosed method.

[0169] The examples illustrate various conditions under which purified casein can be obtained from recombinant *E. coli*. While specific conditions are mentioned in these examples, it is noteworthy that specific combinations of temperature / holding time at that temperature (110°C / 75 sec; 110°C / 30 sec; 120°C / 30 sec; 140°C / 30 sec; 140°C / 2 sec) are disclosed to address technical problems related to casein purification. It is advisable to avoid overheating the composition and to use times longer than extremely short. The various conditions (using heating temperatures of 95°C–140°C and holding times of 2–120 seconds in a heating chamber) all make it possible to obtain casein with suitable concentrations and good purity in the soluble fraction after heat treatment.

[0170] It is recommended to preheat the composition from the first container before performing the heating step within the conduit. This can be done in a chamber outside the first container or in the conduit that guides the composition from the first container to the conduit, where the composition is heated at a desired temperature for a desired duration. The preheating temperature can be 20°C or 30°C lower than the target heating temperature, or about 20°C to about 60°C lower than the heating temperature. Preheating between 75°C and 90°C (e.g., at 80°C) is suitable. The total duration of the treatment (preheating + heating + chamber (held at the heating temperature) + precooling + cooling) is approximately 150-350 seconds, preferably 150-320 seconds, or 200-320 seconds.

[0171] To achieve the desired temperature within the pipe, especially when heating is performed via heat exchange, heating is carried out at a temperature higher than that desired temperature (typically about 5°C higher), taking into account the temperature gradient from the outside of the pipe to its center. Heating can be performed by either of the following methods: Direct steam injection: Steam is injected directly into the pipe to heat it. Steam is generated in a boiler and introduced into the pipe, where it transfers heat to the pipe walls, thereby heating the product flowing through it.

[0172] Indirect heating via heat exchange: using double-walled pipes. The composition flows through the inner pipe, while steam circulates in the space between the two walls. The heat from the steam is transferred through the pipe walls, thus heating the product.

[0173] Electric heating: The heating element is placed inside the pipe to heat the pipe, and the heat is generated directly in the pipe wall by using electrical energy.

[0174] Induction beam heating: This method uses electromagnetic induction through the Joule effect to induce an alternating current in the pipe wall with the help of a magnetic field to heat the pipe.

[0175] In some embodiments, the composition is circulated within the pipe at a flow rate between 15 and 30 L / h.

[0176] The composition is typically cooled and then transferred to a second container after flow. In some embodiments, the soluble fraction recovered by centrifugation and / or filtration after flow and heating is transferred to the second container. Further purification or concentration of casein (e.g., by evaporation) and / or quantification of casein in the soluble fraction can then be performed.

[0177] This method may yield a casein composition wherein casein comprises 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. Specifically, the ratio of casein to total protein in the soluble fraction is increased compared to the ratio of casein to total protein in the microbial composition.

[0178] In summary, the methods disclosed herein enable the preparation of casein compositions with low environmental impact. The readily available (or obtainable) casein compositions prepared by the methods disclosed herein are another subject of this invention. Such compositions can be characterized as defined above.

[0179] In milk, casein exists in micelle form, wherein micelles comprise α-S1 casein, α-S2 casein, β-casein, and κ-casein assembled in identical particles. In the case of this invention, casein may not be assembled in micelle form. However, this does not preclude the ability to obtain curd from the casein composition by adding a suitable curdling agent.

[0180] In fact, as developed in the examples, this casein composition can be used to produce cheese substitutes, particularly animal-free cheese substitutes, i.e. cheese substitutes that do not contain any animal-derived products (especially when the casein is isolated from bacterial or yeast cultures).

[0181] In summary, simple and brief heating (less than 5 minutes, or preferably less than 2 minutes) through a pipe to a composition containing casein and other proteins allows for the enrichment of casein in the composition by reducing the amount of most other proteins in the soluble fraction. Heating also allows for the dissolution of casein present in the insoluble fraction of the bacterial culture. While the resulting casein is not under optimal conditions for micelle formation, it can be appropriately coagulated under the action of a coagulant. This finding is unexpected.

[0182] Specifically, as shown in WO2022253816, curdling can be achieved using recombinant casein produced in bacteria, and this is achieved in the absence of κ casein, although these recombinant caseins do not have post-translational modifications (such as phosphorylation), and κ casein is known to play an important role in the formation of casein micelles in dairy products.

[0183] In one embodiment, a method for obtaining a dairy substitute is disclosed, comprising performing the methods disclosed herein to obtain a casein composition, mixing the casein composition with at least one other ingredient to obtain a liquid pre-curled milk composition (LpCC), and further processing the liquid pre-curled milk composition (LpCC) to obtain a dairy substitute, the other ingredient comprising at least one component selected from proteins, water, calcium, lipids, and carbohydrates. Further processing of the LpCC particularly includes providing a coagulant to the LpCC to obtain curd. The curd can then be drained (the resulting whey can be reused in the methods disclosed herein, thus achieving recycling), shaped, and aged to obtain a cheese substitute. The curd can be obtained and processed using lactic acid bacteria (Streptococcus thermophilus and Lactobacillus bulgaricus) to obtain a yogurt substitute.

[0184] This implementation scheme is disclosed in particular in WO2022058573 and WO2022253816.

[0185] As previously mentioned, the curdling agent is preferably not rennet but an acidifier, especially lactic acid bacteria or lactic acid, citric acid or acetic acid. The casein composition may contain only α-S1 casein (as casein), or only α-S2 casein (as casein), or only β-casein (as casein), or only α-S1 and β-casein (as casein), or only α-S2 and β-casein (as casein), or only α-S1 and α-S2 casein (as casein), or α-S1, α-S2 and β-casein.

[0186] Curd can be processed to obtain edible compositions, particularly cheese substitutes, as disclosed in WO2022058573 and WO2022253816. These two documents are incorporated herein by reference, particularly the sections describing the preparation of LpCC or edible compositions from compositions containing casein (especially those produced in bacteria and / or without κ casein), and especially the sections concerning the preparation of cheese substitutes having the essential characteristics of fresh cheese and cheese substitutes having the essential characteristics of soft or semi-soft cheese. Parts of these documents, particularly WO2022253816 concerning calcium and other salts, lipids, emulsifiers and gelling agents, carbohydrates, vitamins, curdling agents, fermentation products, moisture, and curd processing, are also incorporated by reference.

[0187] The teachings on LpCC compositions (depending on the type of cheese substitute desired) and the amount of gelling agent added (especially WO2022253816, pp. 24-28) are also incorporated by reference.

[0188] It is worth noting that gelling agents (especially agar) can be added together with other ingredients before or after curdling.

[0189] Preferably, all other ingredients added in b) are non-animal derived.

[0190] The ingredients added in ii) include i. optional proteins other than casein, ii. lipids, iii. water, and iv. carbohydrates; in a preferred embodiment, the carbohydrates do not include lactose to make the edible composition more acceptable to customers with lactose intolerance.

[0191] Taking into account water loss due to curdling and aging (which can be adjusted by those skilled in the art by changing the duration and maturation conditions), the selection and amount of ingredients are adjusted so that the LpCC composition is suitable for the final composition desired for an edible composition.

[0192] Specifically, the processing of LpCC includes: vi. adding at least one coagulant to a liquid composition to obtain a curd, and vii. further processing the curd to obtain a dairy substitute, wherein the dairy substitute is a cheese substitute or a yogurt substitute.

[0193] This further step vi) may involve acidic precipitation of casein. Specifically, the acidic precipitation reaction is carried out at a pH between 4 and 5 (e.g., pH=4.6), preferably at room temperature (about 20°C), but can also be carried out at about 90°C. Several other purification steps may be performed.

[0194] Further processing of the curd may include the following steps: a) mixing a structuring agent with the curd to obtain a supplementary curd, wherein the structuring agent is selected from gelling agents, texturers, emulsifiers and mixtures thereof, and b) shaping and draining the supplementary curd.

[0195] Other methods may include: a) providing a casein composition comprising the recombinant casein disclosed herein; b) mixing the casein composition with at least one other ingredient comprising at least one component selected from water, calcium, lipids and carbohydrates to obtain a liquid pre-curled milk composition (LpCC); c) adding at least one curdling agent to the liquid pre-curled milk composition to obtain a curd; and d) further processing the curd (particularly by draining) to obtain an edible composition, wherein a gelling agent (particularly agar) is added in b) and / or d).

[0196] In one implementation, a gelling agent is added in b).

[0197] In another implementation, a gelling agent is added in d).

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

[0199] In this embodiment, it is preferred that the edible composition has: i. a protein content between 2% and 15% (by weight), and ii. a moisture content of more than 80% on a fat-free basis.

[0200] Therefore, this implementation scheme is well-suited for producing fresh cheese substitutes. Further processing of the curd may include shaping the drained curd and / or aging the dried curd.

[0201] Compared to dairy alternatives obtained by the same method (but in which whey is not added to produce casein via fermentation, and in which lactose in whey is replaced by an equal amount of glucose or an equal amount of sucrose (provided in pure form or in molasses), the environmental impact of dairy alternatives or any other dairy alternatives made from casein obtained by the method described herein can be significantly reduced for at least one of the criteria listed above. Specifically, for at least one of the above factors (particularly climate change), the environmental impact is reduced by at least 5%, or at least 10%, or at least 20%, or at least 25%, or at least 30%. Attached Figure Description

[0202] Figure 1: Plasmid structures expressing β-casein and α-S1 casein.

[0203] Figure 2: α-carboxylates in the soluble and insoluble fractions of the pyrolysis solution before and after heat treatment. s1 SDS-PAGE analysis of casein and β-casein. Lane 1: Size markers, Precision Plus Protein™ Unstained Standards (Bio-Rad, 1610363EDU) - Lane 2: Cell suspension before heat treatment, soluble fraction - Lane 3: Lysis buffer after heat treatment (Experiment 1), soluble fraction - Lane 4: Lysis buffer after heat treatment (Experiment 2), soluble fraction - Lane 5: Lysis buffer after heat treatment (Experiment 3), soluble fraction - Lane 6: Lysis buffer after heat treatment (Experiment 4), soluble fraction - Lane 7: Lysis buffer after heat treatment (Experiment 5), soluble fraction - Lane 8: Cell suspension before heat treatment, insoluble fraction - Lane 9: Lysis buffer after heat treatment (Experiment 1), insoluble fraction - Lane 10: Lysis buffer after heat treatment (Experiment 2), insoluble fraction - Lane 11: Lysis buffer after heat treatment (Experiment 3), insoluble fraction - Lane 12: Heat-treated pyrolysis solution (Experiment 4), insoluble fraction - Lane 13: Heat-treated pyrolysis solution (Experiment 5), insoluble fraction.

[0204] Figure 3: α-carboxylates in the soluble and insoluble fractions of the pyrolysis solution before and after heat treatment. s1 SDS-PAGE analysis of casein and β-casein. Analysis of samples diluted 3-fold. For each plot, lane 1: size markers, Precision Plus Protein. TM 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: Lysis buffer after heat treatment, soluble fraction - Lane 5: Lysis buffer after heat treatment, insoluble fraction. Figure A: 110°C, 75 s; B: 110°C, 30 s; C: 120°C, 30 s; D: 140°C, 30 s; E: 140°C, 2 s.

[0205] Figure 4: Monitoring of growth and glucose concentration in culture media under different culture conditions. Growth (A) was monitored by optical density (OD) at 600 nm. Uninduced glucose-containing cultures are represented by dotted lines, IPTG-induced glucose-containing cultures are represented by dashed lines, and cultures containing both glucose and whey are represented by solid lines.

[0206] Figure 5: Casein production analysis by SDS-PAGE under various induction conditions. Samples were prepared as described in Example 4. T1, T2, T3: Total lysates from glucose-containing but uninduced cultures (T1), glucose-containing cultures induced by IPTG (T2), and cultures containing glucose and whey (T3, as described in Example 4). P1, P2, P3: Insoluble fractions (precipitates) from lysates of glucose-containing but uninduced cultures (P1), glucose-containing cultures induced by IPTG (P2), and cultures containing glucose and whey (P3). S1, S2, S3: Soluble fractions (supernatants) from lysates of glucose-containing but uninduced cultures (S1), glucose-containing cultures induced by IPTG (S2), and cultures containing glucose and whey (S3). A: Purified α-S1 casein and α-S2 casein from Sigma, 1 g / L. A: Purified β-casein from Sigma, 1 g / LM: molecular weight marker. Band sizes are shown on the left side of the gel.

[0207] Figure 6: Comparison of the environmental impact of casein production using different raw materials. Glucose was used as the reference baseline (100%).

[0208] Figure 7: SDS pages of clear cell lysates from the samples; (1) BR1 – SO_gal0, (2) BR2 – SO_gal1, (3) BR3 – SO_gal2, (4) BR4 – SO_gal3 whey-free, and (5) BR5 – SO_gal3 whey-containing. Solutions of commercial α-casein (A) and β-casein (B) were used as controls.

[0209] Figure 8: SDS pages of insoluble fractions from lysates of samples; (1) BR1 – SO_gal0, (2) BR2 – SO_gal1, (3) BR3 – SO_gal2, (4) BR4 – SO_gal3 whey-free, and (5) BR5 – SO_gal3 whey-containing. Solutions of commercial α-casein (A) and β-casein (B) were used as controls.

[0210] Figure 9: Concentrations of lactose (A), glucose (B), and galactose (C) in the culture medium. Detailed Implementation

[0211] Example 1: Production of α-S1 casein and β-casein The synthetic genes encoding α-S1 casein and β-casein (associated with natural genes P02662 and P02666, respectively) were modified to remove the signal peptide and the codons were optimized for expression in *E. coli*. The sequences of the proteins encoded by the new synthesis and the sequences of the new synthetic open reading frames (SEQ ID NO: 2 and SEQ ID NO: 4) are shown in the last column of Table 2. These two synthetic open reading frames were cloned together into pET25b+, replacing the NdeI-HindIII fragment of the vector. The two open reading frames are located in the same operon and are controlled by the T7 promoter of pET25b+, wherein (i) both synthetic open reading frames are controlled by the T7 ribosome binding site and terminated by two TAA stop codons, and (ii) the β-casein open reading frame is closer to the promoter, while the α-S1 open reading frame is farther from the promoter (Figure 1). The resulting plasmids were transformed into the BL21(DE3) strain (Novagene). Individual transformed clones were isolated, and each synthesized gene was validated. One clone was used to inoculate LB medium.

[0212] Table 2: Sequences of natural casein (precursor) and related recombinant proteins. In native casein, the signal peptide is indicated in bold. In recombinant casein, methionine residues resulting from cloning into the pET expression system are indicated in bold.

[0213] To produce a batch of α-S1 casein and β-casein, the obtained strains were cultured as follows: Five 1 mL stock solutions of the strain were stored at -80°C in 10% DMSO, thawed, and used to inoculate five 1 L pre-cultures (inoculation rate 0.1% v / v) using resurrection medium Y15 supplemented with 100 µg / mL ampicillin, consisting of yeast extract (15 g / L) and NaCl (5 g / L). The five pre-cultures were cultured in 4 L or 5 L Erlenmeyer flasks at 30°C and 170 rpm (track diameter: 25 mm) for 9 hours. After 9 hours, an optical density of approximately 3 (60 nm) was reached.

[0214] 2.4 L of the first preculture was inoculated into 120 L of medium (2% v / v) in optimized medium supplemented with 100 µg / mL ampicillin and 15 g / L glucose. Growth parameters (oxygen pressure, agitation, pressure, aeration rate, and pH adjustment) were optimized to achieve an optical density (600 nm) in the range of approximately 15.

[0215] This culture was inoculated into a fermenter containing optimized medium supplemented with 100 µg / mL ampicillin, with an initial volume of 1500 L (6% v / v inoculation rate). Growth parameters (glucose feeding, oxygen pressure, agitation, pressure, aeration rate, and pH adjustment) were optimized to achieve an optical density (600 nm) of approximately 90. When the optical density reached 20, IPTG was added to a final concentration of 0.2 mM. This yielded approximately 2 tons of culture medium containing 3.4% dry biomass.

[0216] Example 2: Ultra-high temperature (UHT) thermal bacterial lysis setting to extract and dissolve α-cells from cells at ultra-high temperatures. s1 Schemes involving casein and β-casein.

[0217] Biomass concentration and washing: The culture medium obtained as described in Example 1 was concentrated using a self-cleaning disc centrifuge (centrifugal force: 20000G, temperature < 20°C). 298 kg of concentrated cells were collected.

[0218] The concentrated biomass was then washed three times with permeate water using the same centrifuge. The concentrated biomass was mixed with water until a homogeneous suspension was obtained. The suspension was then separated by centrifugation, and the first fraction of wash water was discarded. This washing process was repeated twice; a precipitate of 239.1 kg of concentrated cells was finally obtained.

[0219] The heat-induced bacterial lysis was followed by resuspending the washed, concentrated biomass in 4.2 volumes of permeate water until a homogeneous suspension was obtained. The resulting suspension was stored at 60 L 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.

[0220] The five fractions are then heated in a pasteurization chamber by monitoring the holding time at a given temperature using a UHT pasteurizer (Pasteurizer, OMVE, HTST / UHT system, HT220-DSI, configuration: tubular heat exchanger, flow rate through the chamber 16.66-27.2 L / h), with a holding time (time in the chamber) of 2-75 seconds. The temperature in the pasteurization chamber is 110-140°C.

[0221] Five different conditions were tested on five bacterial suspensions to determine the optimal and rapid heat treatment that would allow for efficient bacterial lysis, E. coli protein precipitation, and casein dissolution. The different heating temperatures were set slightly higher than those used in the pasteurization chamber to ensure that the required pasteurization temperatures were achieved.

[0222] Experiment 1: Temperature = 110℃, holding time = 75 seconds.

[0223] Experiment 2: Temperature = 110°C, holding time = 30 seconds.

[0224] Experiment 3: Temperature = 120 °C, holding time = 30 seconds.

[0225] Experiment 4: Temperature = 140 °C, holding time = 30 seconds.

[0226] Experiment 5: Temperature = 140℃, holding time = 2 seconds, in a small-scale pilot test.

[0227] In each case, the product temperature is controlled during the heating step and at the pasteurization chamber outlet, while the supply pressure is measured throughout the process.

[0228] To investigate the efficiency of these five different heat treatment methods, samples were collected before and after each experiment and analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) on polyacrylamide gels (4-20% Criterion). TM Analysis was performed on TGXStain-Free Protein Gel (Bio-Rad, 5678093) using Precision Plus Protein. TMUnstained Standards (Bio-Rad, 1610363EDU) are used as markers.

[0229] For each experiment, 1 mL of bacterial suspension was taken before and after heat treatment and centrifuged (13000 rpm, 5 min, RT) to separate soluble and insoluble fractions. The supernatant was separated from the precipitate and mixed with 2X sample buffer at a 1:1 ratio. This buffer consisted of 0.1 M Tris-HCl, 4% v:v SDS, 0.2% w:v bromophenol blue, and 20% v:v glycerol. The cell pellet was mixed with 1 mL of 2% v:v SDS solution until a homogeneous suspension was obtained and then mixed with the aforementioned 2X sample buffer at the same ratio. Each resulting solution was heated at 95°C for 5 min and then loaded into the wells of a polyacrylamide gel using an electrophoresis buffer consisting of Tris-HCl (25 mM), glycine (250 mM), and SDS (0.1% v:v). The gel was then processed using Gel-Doc. TM The gels were visualized using UV light in the EZ Imager (Bio-Rad). Diluted samples (3-fold) were also used for better quantification. The ratio of casein to total protein was determined by analyzing and comparing the intensity of casein bands with other E. coli protein bands using Imagelab software, through each SDS-PAGE gel analysis.

[0230] The results are summarized in Figures 2 and 3, including undiluted (Figure 2) and diluted (Figure 3) samples. Under all tested pasteurization conditions, *E. coli* cells were effectively lysed, and the vast majority of α-cells were lysed. s1 Both casein and β-casein are soluble: The total amount of casein before and after pasteurization is very similar, but these caseins are mainly present in the insoluble fraction before pasteurization (Figure 3, lane 8), while they are present in the soluble fraction after pasteurization (Figure 3, lanes 3-7), indicating that the processing yield is very high (at least >80%).

[0231] In the soluble fraction of the lysate, the ratio of casein to total protein (Figure 3, lane 4 in each figure) remained essentially consistent across the five experiments, ranging from 75% to 77%. This ratio was significantly higher than that in the unlysaturated fraction (Figure 3, lane 3 in each figure), which exhibited numerous additional protein bands, as shown in Figure 3 (lane 3 in each figure). This ratio was even higher than that in the unlysaturated cellular composition (insoluble + soluble), indicating that the soluble fraction of the lysate was more casein-rich than the initial composition.

[0232] Furthermore, in the five trials, α s1The average casein / β-casein ratio was also very similar, ranging from 47% to 53%, similar to the ratio in the initial composition in the cells before treatment, indicating that under the conditions we used, α s1 There was no deviation in the purification of casein and β-casein.

[0233] Example 3: Thermal Lysis of Bacteria As described in WO2022253816, the synthetic genes encoding α-S1 casein, α-S2 casein, and β-casein (associated with natural genes P02662, P02663, and P02666, respectively) were modified to remove the signal peptides. These were cloned into plasmids, and the resulting plasmids were transformed into the BL21(DE3) strain. Individual transformed clones were isolated, and one clone was inoculated onto LB medium for each synthetic gene.

[0234] Cells were lysed using two different protocols, and total extracts and samples from soluble and insoluble fractions were analyzed by SDS-PAGE. Casein in the soluble and insoluble fractions of the cell extracts was monitored using two different protocols. Cells transformed with an empty vector were used as a control.

[0235] Protocol 1: The cell pellet obtained from 100 mL of culture was lysed by resuspending in lysis buffer (50 mM Tris HCl pH 7.5, 1 mg / mL lysozyme, 0.03 mg / mL DNase). The suspension was incubated on ice for 30 min, followed by sonication for 10 sec (10% amplitude, Q Sonica XL-2000). 10 µL of the total fraction was collected for SDS-PAGE analysis. Soluble and insoluble fractions were separated by centrifugation at 3220 g and 4°C for 20 min. The supernatant was collected and 10% glycerol was added for storage. The pellet was further resuspended in 50 mM Tris with 2% SDS and 10% glycerol was added for storage. 10 µL of each fraction was collected for SDS-PAGE.

[0236] Protocol 2: The cell pellet obtained from 100 mL of culture was lysed by resuspending it in Bugbuster (Millipole) containing 0.4% lysinase (Millipore). The mixture was incubated at room temperature for 5 minutes, followed by centrifugation at 3220 g and 4°C for 20 minutes. The supernatant was collected, and 10 µL of the supernatant was reserved for SDS-PAGE analysis. The pellet was further resuspended in 2% SDS, and 10 µL of the suspension (representing the insoluble fraction) was reserved for SDS-PAGE analysis.

[0237] α-S1 casein was present in both the soluble fraction (S) and the insoluble fraction (P), while α-S2 was predominantly present in the insoluble fraction. β-casein was mainly (Scheme 1) or entirely (Scheme 2) present in the insoluble fraction, as is commonly observed when recombinant proteins are overexpressed in *E. coli*. This indicates that casein can be present in inclusion bodies in *E. coli*. The different quantitative results may be due to variations in the solubility of β-casein or the stability of inclusion bodies depending on the scheme. However, recovering casein from the soluble fraction of the extract using these schemes will result in the loss of most or all of the recombinant protein.

[0238] As described above, the effect of heat lysis on casein was tested using recombinant strains. Clones transformed with empty vectors were used as controls.

[0239] The culture was centrifuged, and the cell pellet was resuspended in 1 volume of sterile water, centrifuged again, washed with another volume of water, and then resuspended in 1 volume of water. Cell lysis was induced by treating 1 mL samples of this cell suspension at 95°C for 0 min (no heating), 10 min, 20 min, 30 min, 60 min, 90 min, and 120 min, followed by centrifugation to separate the soluble and insoluble fractions. 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 the soluble and insoluble fractions were analyzed by SDS-PAGE. Under these conditions, 10 µL of the soluble fraction and 10 µL of the insoluble fraction represent approximately the same amount of total cell extract. Without heating, no cell lysis occurred, or only residual cell lysis occurred in the water. The precipitate contained essentially intact cells, including casein, and the sample effectively represented the total cell extract.

[0240] It has been confirmed that temperature cleavage affects the distribution of casein in both soluble and insoluble fractions.

[0241] When heated at 95°C for 10 minutes (or before), α-S1 casein was almost entirely present in the soluble fraction, while without heating, α-S1 casein was present in similar amounts in both the soluble and insoluble fractions.

[0242] Casein β gradually transfers to the soluble fraction, which contains a large amount of this recombinant protein upon heating for 10 minutes (or before), and contains most of this recombinant protein upon heating for 30 minutes. Without heating, β-casein is mostly or completely present in the insoluble fraction.

[0243] The distribution of casein α-S2 (completely present in the insoluble fraction without heating) appears to be relatively heat-insensitive, remaining largely present in the insoluble fraction even after heating for 120 minutes. However, casein α-S2 appears in the soluble fraction upon heating at 95°C for 20 minutes, and its presence in the insoluble fraction decreases slightly over time.

[0244] Over time, many other protein bands from the insoluble fraction disappeared but did not transfer to the soluble fraction. In the soluble fraction of the heated sample, only some faint protein bands were visible, in addition to the casein band.

[0245] These results indicate that thermal lysis is a good method for the rapid purification of casein, especially α-S1 casein and β-casein.

[0246] Example 4: Production of recombinant casein-based whey as a raw material in a 1L fermenter, used in combination with glucose. To generate whey, semi-skimmed pasteurized 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 to induce curdling. The curd was drained for 48 hours to recover whey, yielding 0.77 kg of whey per liter of milk at pH 4.6 and a lactose concentration of 51.3 g / L. This whey was then filtered through a 0.22 mM filter and autoclaved. SDS-PAGE analysis showed no residual casein in the whey.

[0247] A 1-liter fermenter (Multifors 2 Microbial 1.4LTV, Infors) was inoculated with a culture of the recombinant strain described above, with whey and glucose as carbohydrate sources.

[0248] The medium was supplemented with 50% whey. 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.

[0249] Inoculate 0.5 L of culture medium with a pre-culture of the recombinant clone to achieve an initial OD of 0.25. 600 Fermentation was carried out in a fed-batch manner with pH = 7.2, T = 37°C, and pO2 = 15%. The culture was fed with a solution containing 600 g / L of D-glucose. Ampicillin (100 µg / ml of culture) was added after 14 hours.

[0250] Lactose can induce casein expression from the pET carrier in BL21. However, at the start of fermentation, glucose inhibits lactose uptake through carbon catabolism inhibition (Görke, B., Stülke, J. Carbon cataboliterepression 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 fermentation, only glucose is consumed.

[0251] Approximately 14 hours later, OD 600 =40 (Figure 4), interrupting glucose feeding for 30 minutes caused a sharp drop in glucose concentration in the culture medium, enabling metabolic conversion and inducing lactose consumption and casein expression. After 30 minutes, glucose feeding was resumed, but at a reduced rate to allow the culture to grow without hindering lactose consumption. In this respect, this induction method differs from that of Viitanen et al. (Viitanen et al., 2003, Cheese whey-induced high-cell-density production of recombinant proteins in Escherichia coli, MicrobialCell Factories 2:2); in this study, induction was achieved by adding whey to a glucose-free medium supplemented with glycerol. Culture was stopped at T=21 hours, where OD 600 =100.7. At this point, no lactose was detected in the culture medium. Cells were harvested by centrifugation at room temperature.

[0252] As a negative control, the same culture medium without whey (replaced with water) was used, with all other components at the same final concentration. Therefore, glucose was the sole carbohydrate source, and no casein expression was expected. Culture was stopped at T=21 hours, where OD... 600=94.6. Cells were harvested by centrifugation at room temperature. As a positive control for casein expression, the same medium without whey (using water instead) was used, and induction was achieved by adding isopropyl β-D-1-thiogalactoside (IPTG) (a lactose analog whose induced effect cannot be inhibited by glucose) at OD=40. Culture was stopped at T=21 hours, where OD 600 =80.0. Cells were harvested by centrifugation at room temperature. Conditions are summarized in Table 3.

[0253] Table 3. Summary of culture conditions and induction mechanisms under different conditions. For analysis, equal cell aliquots were centrifuged and resuspended in 1 / 2 volume of sterile water. A 1 ml sample of this cell suspension was heated at 95°C for 60 minutes, as described previously (PCT / EP2022 / 064728), resulting in cell lysis, casein lysis, and degradation and / or precipitation of other proteins in the soluble fraction. The soluble and insoluble fractions were then 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.

[0254] Purified β-casein from milk (Sigma) was used as a control, but it showed 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).

[0255] As shown in Figure 5, casein expression was effectively induced by IPTG in the presence of glucose and in whey-containing media. Casein production was highly efficient when using whey. Recombinant casein yields were estimated to be in the range of 2.5 g / L (glucose conditions) and 5 g / L (whey conditions), when normalized to cell density (see OD above). 600 The whey conditions used are twice as effective as the glucose conditions, and twice as effective.

[0256] The amount of sugar consumed was estimated in all samples. Under the conditions used, *E. coli* BL21 was able to take up and metabolize glucose. Even at low glucose concentrations, *E. coli* was able to take up lactose and hydrolyze it into glucose and galactose; although galactose consumption was minimal, glucose derived from lactose was metabolized very efficiently. Therefore, glucose consumption was estimated to be the difference between the glucose added to the culture medium and the glucose produced from lactose hydrolysis. The estimate was based on the mass of added sugar, as the glucose concentration in the culture medium was negligible at the end of the experiment.

[0257] The results are shown in Table 4. As the table shows, both the added glucose and glucose from lactose hydrolysis were effectively consumed during fermentation, although glucose consumption from the whey (lactose source) was extremely low before the glucose concentration decreased. Under the conditions we used, glucose from lactose hydrolysis accounted for 12% of total glucose consumption, indicating that lactose contributes to the carbohydrate feedstock. Galactose from lactose hydrolysis was not consumed.

[0258] Table 4: Sugar Consumption Example 5: Environmental Impact. The environmental impact of casein obtained from bacterial fermentation using various sugars was quantified using a multi-standard 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).

[0259] The Life Cycle Inventory (LCI) is based on the average of raw data from laboratory and pilot-scale experiments. Data not collected directly from experimental results is from industrial projects based on production processes, particularly in utilities and processes, or from their partners.

[0260] The effects of various sugar sources were tested, and glucose was considered to be derived from corn (European origin), sucrose was considered to be derived from sugar beets (other parts of the world, excluding Switzerland), molasses was considered to be derived from sugar beets (other parts of the world, and was considered to contain 50% sugar by weight), and whey was considered to be derived from cheese manufacturing processes (global origin).

[0261] We assume that the difference lies only in the environmental impact of the raw materials, and that changes in the raw materials will not affect other parameters.

[0262] The LCI for each sugar ingredient was calculated using the Ecoinvent 3.9 database based on its origin.

[0263] Compared to Example 4, the differences are as follows: - This strain is considered gal+, i.e., possessing a highly efficient Leloir pathway and capable of consuming the galactose portion of lactose; - When using sucrose or molasses, this strain is capable of consuming sucrose, as described above; - The sugar fed to this strain contains 100% glucose or 68.6% glucose and 31.4% of another sugar, which can be sucrose, sucrose from molasses, or lactose from whey; - The fermentation tank is 350 liters; - A portion of the water in the process is recycled.

[0264] The purification process includes the following steps: - recovering biomass and washing it three times as described in Example 2, - heating the washed biomass at 95°C for one hour, - recovering the supernatant, - precipitating casein with HCl at pH 4.6 and washing the precipitate, - resuspending the acidic casein precipitate and neutralizing it with sodium hydroxide (pH=7), - drying sodium caseinate by spray drying to reduce the moisture content to 5%.

[0265] The Life Cycle Impact Assessment (LCIA) was conducted using datasets from the Ecoinvent 3.9 database (Zurich, Switzerland) and Agrybalise (Ademe, France). All impacts were attributed to the casein composition, not to byproducts of the process, such as biomass. The sour whey used as a carbon feedstock could be considered either waste from soft cheese production or a value-added byproduct. Therefore, in the first case, the use of whey has no environmental impact. In the second case, the use of whey is accounted for as emissions from the final product based on the EF 3.1 dataset in Ecoinvent 3.9.

[0266] The results of the 16 standards (unit definitions are above) are expressed as relative values ​​when glucose is the only raw material, as shown in Figure 6.

[0267] When whey is treated as waste, the environmental impact of all 16 criteria is significantly lower than that of glucose, and for all criteria except the following, it is lower than or equal to the impact of other sugars: - Compared to molasses, number ix (human toxicity: non-carcinogenic), because beet derivatives have the property of fixing heavy metals in the sucrose production process.

[0268] - Compared to sucrose, it is numbered v, ix, and the slightly lesser type is numbered iii.

[0269] For the value-added aspects of whey, the environmental impact on land use standards is higher than that of other sugars because livestock farming contributes an impact equivalent to large-scale land use, which in this case is attributed to the raw material. However, it is: - lower than glucose on all other standards, - lower than sucrose on 11 of the 16 standards (i, ii, iv, vi, vii, x, xii, xiii, xiv, xv, xvi), and - lower than molasses on 8 of the 16 standards (ii, v, vi, vii, x, xiv, xv, xvi).

[0270] In summary, using whey as a carbon source in processes for obtaining recombinant casein from microbial sources has a better overall environmental impact than when using other carbon sources, for a standard of 50% or more.

[0271] Example 6: Construction of a strain for consuming lactose and galactose. The *E. coli* BL21(DE3) strain was constructed to consume glucose and some lactose (but not galactose derived from lactose). A new *E. coli* BL21(DE3) strain was generated to express the galETKM operon from the *E. coli* K12 strain, which is essential for galactose consumption. The galETKM operon contains four genes: galE encodes an epimerase, galT encodes uridyltransferase, galK encodes galactokinase, and galM promotes the interconversion between β-galactose and α-galactose.

[0272] The constructed BL21(DE3) strain carries gal operons under different promoters, located on low-copy plasmids (pSO148 and pSO149) or in the chromosome (see sequences, Table 5).

[0273] Table 5: Genetic constructs encoding the galactose consumption pathway Example 7: Production of Recombinant Casein in a 1-L Fermenter Using a Lactose- and Galactose-Consuming Strain Casein production was tested using the BL21 strain, which is capable of consuming both lactose and galactose, with whey and glucose conjugated as feedstock. Acid whey was generated as described in Example 4 and then centrifuged at 12,000 rpm and 4°C for 20 minutes. The supernatant was then filtered through a 0.22 μM filter.

[0274] Cultures of each recombinant strain described in Table 6 were used to inoculate a 1-liter fermenter (Multifors 2 Microbial 1.4L TV, Infors) containing whey and glucose as the sole carbohydrate source.

[0275] Table 6: Strains used in this experiment. The medium used was MSA (medium-weighted agar) containing 50% whey, 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 solution (Struktol), and trace elements. This 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 the SO₂gal1 and SO₂gal2 cultures to a final concentration of 100 µg / mL. The lactose concentration in this medium was approximately 20 g / L. For cultures using SO_gal3, a minimal culture medium (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% antifoam solution (Struktol)) and trace elements were used, containing 20 g / L lactose. Conditions are summarized in Table 7.

[0276] Table 7: Fermentation Conditions Inoculate 0.5 L of culture medium with a pre-culture of the recombinant clone to achieve an initial OD of 0.25. 600nm Fermentation was carried out in a fed-batch mode at pH 7.2, T 37°C, and pO2 15%, with the culture fed a solution containing 600 g / L D-glucose. Ampicillin (100 µg / ml culture) was added after 12.5 hours.

[0277] During the growth phase (t=0 to t=10 hours), glucose inhibits lactose uptake through carbon catabolism inhibition, consuming only glucose. Under our culture conditions, glucose was consumed within 10 to 12 hours, and no additional glucose was added for at least 30 minutes to achieve a metabolic switch to lactose consumption, thereby inducing casein expression. This switch was correlated with a rapid increase in pO2 concentration in the bioreactor. Culture was stopped at T=17.8 hours, at which point OD... 600 The range is between 94 and 107.

[0278] For analysis, aliquots of cells were centrifuged and resuspended in a 1:1 volume of lysis buffer (Tris HCl, pH 8.5). A 2 mL sample of this cell suspension was processed by heating at 95°C for 60 minutes, as described in Example 4. 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. As shown in Figures 7 and 8, casein expression was effectively induced under all conditions. The yield of recombinant casein was estimated to be in the range of 2.5 to 6 g / L in the bacterial culture.

[0279] Lactose, glucose, and galactose consumption were measured in all samples. The results are shown in Figure 8. As these figures indicate, lactose was completely hydrolyzed, and both the glucose added during fermentation and the glucose produced from lactose hydrolysis were effectively consumed. Galactose from lactose hydrolysis was consumed in BR2, 3, 4, and 5, while galactose accumulated in BR1. As expected, strains SO_gal1, SO_gal2, and SO_gal3 could consume galactose, while SO_gal0 could not.

Claims

1. A method for obtaining a casein composition, comprising: i. Providing microorganisms transformed with at least one nucleic acid encoding casein; ii. Culturing said microorganisms in a whey-containing medium to express and produce casein; iii. thereby providing a microbial composition wherein the pH of said composition is equal to or higher than 6.5, and preferably lower than 9; iv. Heating said microbial composition to reduce the amount of other proteins in the soluble fraction of said composition, wherein heating is carried out at a temperature equal to or higher than 75°C; v. Recovering the soluble fraction from the heated cell composition of iv) thereby obtaining a casein composition in said soluble fraction.

2. The method of 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 exponential or stationary phase.

4. The method according to any one of claims 1 to 3, wherein in addition to whey, another carbon source is present in the culture medium in ii, wherein this other source of the culture medium is preferably sugar, especially glucose, fructose or sucrose which may be present in molasses.

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

6. The method according to claim 5, wherein the bacteria is Escherichia coli, particularly gal+ Escherichia 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 of claim 7, wherein the at least one nucleic acid is under the control of a promoter fused to the laco operon region of the lactose operon, wherein the microorganism expresses the lacI repressor protein of the operon.

9. The method according to any one of claims 1 to 8, wherein the culture of said microorganism is initiated in the presence of sugars other than lactose, and wherein the consumption of lactose present in whey begins during the exponential growth phase, particularly near or after the middle of the exponential phase of said microorganism, or at the beginning of the stationary growth phase.

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% (w / v).

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

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

13. The method according to any one of claims 1 to 12, wherein lactose accounts for at least 2% (w / w, 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 prior to 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 lysing the microbial composition prior to (iv).

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

17. The method according to any one of claims 1 to 16, comprising further processing the soluble fraction by adding at least one step selected from the steps of adding 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 the casein composition is performed at a temperature equal to or higher than 95°C.

19. The method according to any one of claims 1 to 18, wherein the casein is selected from β-casein, α-S1 casein, α-S2 casein and mixtures thereof, particularly wherein the casein is selected from β-casein, α-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 α-casein and another nucleic acid encoding β-casein, wherein the expression of both nucleic acids is induced by lactose.

21. The method according to any one of claims 1 to 20, wherein the microorganism is not converted with nucleic acid encoding κ casein.

22. The method according to any one of claims 1 to 21, wherein the casein comprises 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 ratio of casein to total protein in the soluble fraction is increased relative to the ratio of casein to total protein in the microbial composition.

24. A method for culturing microorganisms, comprising: i. Providing 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 medium containing glucose as a carbon source, wherein the expression of the nucleic acid is not induced; iii. Depleting the glucose in the medium when the microorganism is in the exponential growth phase, wherein whey is present in the medium at the time of depletion, thereby providing lactose present in the whey as a carbon source, and wherein the expression of the nucleic acid is induced by the lactose present in the whey; iv. Further culturing the microorganism in the presence of the carbon source.

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

26. The method of claim 24, wherein whey is not present as a carbon source in IV.

27. The method of claim 24, wherein glucose and whey are carbon sources in IV.

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 casein is under the control of a promoter fused to the laco operon region of the lactose operon, wherein the microorganism expresses the lacI repressor protein 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, but is added in iii.

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

33. A method for reducing the environmental impact of a casein composition prepared by fermentation of genetically modified microorganisms, comprising: i. Providing microorganisms transformed with at least one nucleic acid encoding casein; ii. Culturing said microorganisms in a whey-containing medium to express and produce casein; iii. thereby providing a microbial composition wherein the pH of said composition is equal to or higher than 6.5, and preferably lower than 9; iv. Heating said microbial composition to reduce the amount of other proteins in the soluble fraction of said composition, wherein heating is carried out at a temperature equal to or higher than 75°C; v. Recovering the soluble fraction from the heated cell composition of iv), thereby obtaining a casein composition from said soluble fraction, wherein the environmental impact is reduced compared to the environmental impact of a casein composition obtained by the same steps except that glucose or sucrose or molasses is used instead of whey in step b.

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

35. A method for obtaining a dairy substitute, comprising performing the method of any one of claims 1 to 23, mixing a casein composition with at least one other ingredient to obtain a liquid pre-cured milk composition (LpCC), and further processing the liquid pre-cured milk composition (LpCC) to obtain a dairy substitute, wherein the at least one other ingredient comprises at least one component selected from proteins, water, calcium, lipids, and carbohydrates.

36. The method of claim 35, wherein processing the LpCC comprises: vi. Adding at least one curdling agent to a liquid composition to obtain curd, and vii. Further processing the curd to obtain a dairy substitute, wherein the dairy substitute is a cheese substitute or a yogurt substitute.

37. The method of claim 36, wherein step vi) further comprises heating at room temperature or 90°C and performing acid precipitation of casein at pH 4.

6.

38. A dairy product substitute that is readily obtainable by the method of any one of claims 35 to 37.

39. The dairy substitute of claim 38, wherein the environmental impact is reduced for at least one criterion listed in claim 34.

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