METHOD FOR PRODUCING CHEESE SUBSTITUTES
20230337692 · 2023-10-26
Inventors
Cpc classification
International classification
Abstract
The invention relates to a method for the production of cheese substitutes comprising mixing a casein composition comprising only casein of non animal origin with other ingredients comprising at least one ingredient among water, calcium, lipids, and carbohydrates, so as to obtain a liquid composition, transforming the liquid composition into a curd by the addition of at least one curdling agent and further processing the curd to obtain a cheese substitute.
Claims
1. A method for producing an edible composition comprising: (a) providing a casein composition comprising only casein of non-animal origin; (b) mixing the casein composition with at least one other ingredient selected from water, calcium, lipids, and carbohydrates to obtain a liquid pre-curd composition (LpCC), (c) adding at least one curdling agent to the liquid pre-curd composition to obtain a curd, and (d) further processing the curd to obtain an edible composition.
2. The method of claim 1, wherein a gelling agent is added in (b) or in (d).
3. The method of claim 1, wherein the at least one other ingredient added in (b) is of non-animal origin.
4. The method of claim 1, wherein the at least one other ingredient added in (b) comprises: (i) optionally, proteins other than caseins, (ii) lipids, (iii) water, and (iv) carbohydrates that do not comprise lactose.
5. The method of claim 1, wherein the curdling agent does not comprise any agent of animal origin, and comprises an acidifying agent, the acidifying agent being an acid or a ferment.
6. The method of claim 1, wherein a ferment for maturation is added in (c) together with the curdling agent.
7. The method of claim 1, wherein the edible composition comprises: (i) a protein content between 2 and 25% (w/w), and (ii) a moisture content above 62%, on a fat-free basis.
8. The method of claim 1, wherein the casein composition does not contain at least one casein selected from alpha-S1 casein, alpha-S2 casein, beta casein, and kappa casein.
9. The method of claim 8, wherein the casein composition does not contain kappa casein.
10. The method of claim 9, wherein the casein composition contains only alpha-S1 casein, only beta-casein, or a mixture of alpha-S1 casein and beta-casein.
11. The method of claim 5, wherein the acidifying agent comprises a lactic bacteria.
12. The method of claim 1, wherein an emulsifying agent is added in (b) or (c) before curdling.
13. The method of claim 1, wherein a gelling agent is added in the LpCC.
14. The method of claim 13, wherein the gelling agent is agar-agar.
15. The method of claim 13, wherein the gelling agent is locust bean gum.
16. The method of claim 1, wherein the edible composition comprises: (i) a protein content between 15 and 25% (weight content), and (ii) a moisture content between 62% and 80%, on a fat-free basis.
17. The method of claim 16, wherein the LpCC obtained in (b) comprises: (i) a protein content between 10 to 18% (weight content), and (ii) a moisture content above 62%, on a fat-free basis.
18. The method of claim 16, wherein agar-agar is added to the LpCC in an amount of 0.5% to 1% (w/w) of the LpCC.
19. The method of claim 16, wherein a ferment for maturation is added in (c), and wherein (d) comprises salting and molding the curd.
20. The method of claim 19, wherein (d) comprises maturing the curd at a temperature allowing for the development of the ferment for maturation.
21. The method of claim 16, wherein the edible composition has a lipid content between 10 and 30% (weight content).
22. The method of claim 16, wherein the LpCC has a lipid content between 10% and 30% (weight content).
23. The method of claim 16, wherein the edible composition has a calcium content below 2.8% (weight content).
24. The method of claim 16, wherein the LpCC has a calcium content between 0.3 and 1.7% (weight content).
25. The method of claim 1, wherein the edible composition comprises: (i) a protein content between 2 and 15% (weight content), and (ii) a moisture content above 80%, on a fat-free basis.
26. The method of claim 25, wherein the edible composition has a protein content between 5 and 15% (weight content).
27. The method of claim 25, wherein the LpCC obtained in (b) comprises: (i) a protein content below 12% (weight content), and (ii) a moisture content above 80%, on a fat-free basis.
28. The method of claim 27, wherein the LpCC obtained in (b) comprises: (i) a protein content between 5% and 10%% (weight content), and (ii) a moisture content above 80%, on a fat-free basis.
29. The method of claim 25, wherein agar-agar is added to the LpCC in an amount of 0.5% to 1.0% (w/w) of LpCC.
30. The method of claim 25, wherein the edible composition has a lipid content between 10% and 30% (weight content).
31. The method of claim 25, wherein the LpCC has a lipid content between 10% and 30% (weight content).
32. The method of claim 25, wherein the edible composition has a calcium content lower than 2.8% (weight content).
33. The method of claim 25, wherein the LpCC has a calcium content between 0.1% to 1% (weight content).
34. The method of any of claim 25, wherein further processing the curd comprises: (a) mixing a gelling agent with the curd to obtain a supplemented curd, and (b) molding and draining the supplemented curd.
Description
DESCRIPTION OF DRAWINGS
[0344]
[0345]
[0346]
[0347]
[0348]
[0349]
[0350]
[0351]
[0352]
EXAMPLES
Example 1: Making of a Soft Cheese Substitute
Obtaining a Casein Composition: Mixing Casein, Calcium and Water:
[0353] 90 g of casein composition (Slow-Release Casein, MyProtein) were mixed with 10 g of calcium carbonate (Calcidose Mayoly Spinder) and 500 ml water. MyProtein Slow-Release casein is a dry composition comprising, for 100 g total: 76 g of proteins (caseins), 1.5 g of lipids, 4 g of glucids, and 0.25 g of salts. The mixture was incubated for more than 2 hours at a temperature between 4° C. and 8° C., for homogeneization.
Preparation of the Liquid Pre-Curd Composition
[0354] The casein composition obtained above was heated at 35° C. Then, 3 g of glucose (Dextrose Sugar, ToquedeChef, France) and 40 g of deodorised coconut oil (BioPlanète, France), or sunflower oil (Monoprix, France) were added and the solution was mixed. Protein content in the LpCC was estimated to be about 10% (weight content).
Curdling
[0355] 0.5 g of Beaugel 9 (Etablissement Coquard, France) mixture containing rennet, lactic ferments and ripening ferments were added and the composition is incubated at 20° C. for two to twelve hours, in a mold, and then at low temperature (between 4° C. and 8° C.) for 16 hours. Composition’s weight was measured in order to precisely monitor draining in the further steps.
Draining, Salting and Aging
[0356] After 16 hours at low temperature, the product was removed from the mold, and placed into a draining mold for draining. The cheese mold was placed into a box to control hygrometry. About ¼ to ½ teaspoon of table salt (La Baleine, France), depending on desired taste, can be spread on the upper face and the product is incubated at 14° C. Twenty-four hours later, the product is flipped, and the same salt composition is spread on the other face. The product is then removed from the draining mold, set on a grid and incubated at 14° C. into the refining box to control hygrometry, and flipped every second day, for several weeks. During this period, water is removed from the cheese mold on a regular manner.
[0357] Product’s weight is monitored every day, in order to evaluate the level of draining. It is considered that weight loss consists essentially in water. Since the initial composition is known, an evaluation of water loss allows for the calculation of the product composition, in terms of moisture and protein composition at a given time. Aging is stopped after the product has reached the characteristics of a soft cheese (notably <80% moisture, fat-free basis), but before it has the low moisture of a firm cheese (<62%, fat-free basis).
[0358] A 2-week-old product is featured on
Testing
[0359] The product was evaluated for aspect, color, smell and taste by a panel of 12 person, and found to have the aspect, smell and taste of a soft cheese.
[0360] Protein content in the LpCC was estimated to be about 10% (weight content). Tries with lower amounts of proteins proved to be difficult for a soft cheese, resulting in less solid textures. It could be estimated however, that protein composition could be increased easily. However, by 25% of protein in LpCC, one can reasonably infer that any substantial draining (>10% of loss of total weight) would result in a protein content closer to a firm cheese. In order to allow more than 30% of loss of total weight, we infer that in LpCC, a protein content inferior to 18% (weight content) is preferable.
Example 2: Making of a Fresh Cheese Substitute
Obtaining a Casein Composition: Mixing Casein, Calcium and Water:
[0361] 8 g of micellar casein (Slow-Release Casein, MyProtein,) were mixed with 1 g of calcium carbonate (Calcidose Mayoly Spinder) and 190 ml or 250 ml water. The mixture was incubated for more than 2 hours at a temperature between 4° C. and 8° C., for homogeneization. The composition is then mixed with 12 g of a deodorised coconut oil (BioPlanète, France), or sunflower oil (Monoprix, France).
Preparation of the Liquid Pre-Curd Composition
[0362] The casein composition was heated at 35° C. Then, 12.5 g of glucose (Dextrose Sugar, ToquedeChef, France) were added. pH was closed to 6.5.
Curdling
[0363] 0.5 g lactic ferments (Streptococcus thermophilus, Lactobacillus bulgaricus, Mon yaourt maison, Alsa) were added and the composition was incubated at 40° C. to 45° C. for 8 to 10 hours. pH is then close to 4.
[0364] After this step, a cheesecloth is used to remove some liquid to obtain a more compact composition.
Mixing With Salt and Texturing Agents
[0365] Texturing agents (Xanthan gum, Carob bean gum, Guar gum, Arabic gum, ...) can be added if necessary.
[0366] After 12 hours at low temperature (between 4° C. and 8° C.), the product can be consumed.
[0367] Product’s weight is monitored, in order to evaluate the level of draining. It is considered that weight loss consists essentially in water. Since the initial composition is known, an evaluation of water loss allows for the calculation of the product composition, in terms of moisture and protein composition at a given time, in order to check that it has the properties of a fresh cheese (notably >80% of moisture, fat-free basis).
[0368] A fresh product is featured on
Testing
[0369] The product was evaluated for aspect, color, smell and taste by a panel of 10 persons, and found to have the aspect, smell and taste of a fresh cheese.
[0370] Protein content in the LpCC was estimated to be about 2% (weight content) when 250 ml water were added in the LpCC and about 3% (2.7%) when 210 ml water were added in the LpCC. Protein content could be increased easily. However, by 12% of protein in LpCC, one can reasonably infer that any substantial draining (>20% of loss of total weight) would result in protein composition and a moisture content close to a soft cheese. In order to allow more than 20% of loss of total weight, we infer that in LpCC, a protein content inferior to 10% (weight content) is preferable.
[0371] Tries with protein contents lower than 2% proved to be possible but to result in semi-solid textures, unless extensive draining was used. We infer that a protein content superior to 2% (weight content) is preferable. In addition, in order to make fresh cheeses with high protein contents, with low draining and low water wasting, a protein content in the LpCC superior to 5% (weight content) would be even preferable.
Example 3: Making of a Cheese Substitute With Calcium Caseinate
Obtaining a LpCC: Mixing Calcium Caseinate, Carob Powder, Water, Deodorized Coconut Oil and Glucose:
[0372] A quarter of a teaspoon of carob powder (Locust bean powder, Top Cake) was mixed with 190 ml of room temperature water. [0373] This solution was heated until boiling and then cooled at room temperature. [0374] - 45 g of calcium caseinate casein (Armor Protéines), 75 g of deodorized coconut oil (BioPlanète, France) and 17 g of glucose were mixed with the cooled previous carob solution using a blender.
[0375] pH was close to 6.5.
Curdling
[0376] 0.5 g lactic ferments (Streptococcus thermophilus, Lactobacillus bulgaricus, mon yaourt maison, Alsa) were added and the composition was incubated at 40° C. to 45° C. for 10 to 12 hours. pH is then close to 4.5.
[0377] After this step, a cheesecloth is used to remove some liquid to obtain a more compact composition. Incubation at 4° C. for 24 hours inside the cheesecloth.
Mixing With Salt and Texturing Agents
[0378] Texturing agents (Xanthan gum, Carob bean gum, Guar gum, Arabic gum,...) can be added if necessary. Salt, lemon juice and herbs can also be added if necessary.
[0379] After 12 hours at low temperature (between 4° C. and 8° C.), the product can be consumed.
[0380] Product’s weight is monitored, in order to evaluate the level of draining. It is considered that weight loss consists essentially in water. Since the initial composition is known, an evaluation of water loss allows for the calculation of the product composition, in terms of moisture and protein composition at a given time, to check whether it has the properties of a fresh cheese (notably >80% of moisture, fat-free basis). Moisture content may be adjusted by addition of water if necessary.
Testing
[0381] The aspect, smell and taste of the resulting product were that of a fresh cheese.
Example 4: Making of a Soft Cheese Substitute With Calcium Caseinate
Obtaining a LpCC From Calcium Caseinate, N Two Steps:
[0382] Preparing a first composition by mixing calcium caseinate, sunflower oil, carob powder and water [0383] Half a teaspoon of carob powder (Locust bean powder, Top Cake) was mixed with 150 ml of water at room temperature. [0384] This solution was heated until boiling and then cooled at room temperature. [0385] - 50 g of calcium caseinate casein (Armor Protéines) and 30 g of sunflower oil were mixed with the cooled previous carob solution. The mixture was incubated at least 12 hours at a temperature between 4° C. and 8° C., for homogenization.
Obtaining Hydrated Cashew Nuts
[0386] 55 g of non-salted cashew nuts were hydrated overnight at 4° C.
[0387] Then, water was replaced with fresh water, and cashew nuts were boiled for 30 minutes.
Preparation of the Liquid Pre-Curd Composition (LpCC)
[0388] The first composition obtained above was mixed with 67 g of boiled cashew nuts, 40 g of sunflower oil and 20 g of water using a blender.
Curdling
[0389] 1/16 of teaspoon of thermophilic starter cultures (St. thermophilus, Lb. Helveticus, Cashewbert, cheese starter culture) and 1/16 of teaspoon of traditional strain of P. candidum (Cashewbert) were added to the liquid pre-curd composition.
[0390] The composition is incubated at 20° C. for 12 hours, in a mold, and then at low temperature (between 4° C. and 8° C.) for 12 hours. Composition’s weight was measured in order to precisely monitor draining in the further steps.
Draining, Salting and Aging
[0391] After 12 hours at low temperature, the product was removed from the mold, and placed into a draining mold for draining. The cheese mold was placed into a box to control hygrometry. About ¼ to ½ teaspoon of table salt (La Baleine, France), depending on desired taste, can be spread on the upper face and the product is incubated at 12° C. Twenty-four hours later, the product is flipped, and the same salt composition is spread on the other face. The product is then removed from the draining mold, set on a grid and incubated at 12° C. into the refining box to control hygrometry, and flipped every second day, for several weeks. During this period, water is removed from the cheese mold on a regular manner.
[0392]
[0393] Product’s weight can easily be monitored every day, to evaluate the level of draining. Weight loss consists essentially in water. Since the initial composition is known, (with the composition of dry cashew nut being about 22% of carbohydrates, 20% of proteins, and 53% of lipids, and a few percent of water), an evaluation of water loss can be used for the calculation of the product composition, notably in terms of moisture, and achieving the expected composition or a soft cheese, notably in terms of moisture (between 62% and 80% of moisture, on a fat free basis).
Testing
[0394] The product was evaluated for aspect, color, smell and taste by a panel of 4 persons, and found to have the aspect, smell and taste of a soft cheese.
Example 5: Making of a Soft Cheese Substitute With Caseinates Using a Gelling Agent
[0395] A number of variations were made around the procedure described in example 4, and it was observed that in the absence of any texturing agent, the product can be sagging after removing the mold and during the beginning of aging, which was fully corrected in the presence of a gelling agent. One example is given below.
[0396] 50 g of calcium caseinate casein (Armor Protéines, containing 92% caseins; 1% lipids, and 1% calcium) are hydrated overnight with 140 g of water. Cashew nuts (Naturalia; containing 17% proteins, 55% lipids, 12% carbohydrates, 0.02% ashes) are boiled for 30 minutes, and soaked overnight in water, resulting in a 55% weight increase. Composition of hydrated cashew nuts is estimated as follow: 11% proteins, 15% lipids, 8% carbohydrates, 0.01% of salts and 46% water. The day after, 180 g of the hydrated casein composition is mixed with 17.5 g of the hydrated cashew nuts, 51 g of sunflower oil and 7.5 g of glucose, for a total weight of 256 g. This composition is then pre-heated at 45-50° C., and 250 g are mixed with 50 g of a hot agar-agar/water boiled preparation (4 to 6% agar-agar), to achieve an agar-agar concentration in the range of 0.66 to 1%. LpCC composition is described in Table 2 for 0.66% agar-agar (For other percentages, agar-agar and water contribution should be adjusted).
[0397] Curdling, Draining, salting, aging, weight monitoring and testing were performed as described in Examples 1 and 4, using a variety of ferments, except that curdling ferments were added shortly after the addition of agar-agar, and the composition was let for 12 h at 20° C., and then for 48 h at 14° C., before to be removed from the mold, and placed on grid in a refining box. To achieve the desired moisture, (between 62% and 80% of moisture, on a fat free basis) final product should not have a weight less than 66% of initial.
[0398] Products were matured 7 to 10 days. The products were evaluated for aspect, color, smell, and taste by a panel of 4 persons, and found to have the aspect, smell and taste of a soft cheese. The characteristics of soft cheese were preserved, in terms of moisture and proteins content (15% to 25% of total weight). Product can then be wrapped and stored at low temperature (between 4° C. and 14° C.).
TABLE-US-00003 Composition of the LpCC Ingredient in grammes in% proteins 44.4 g 14.8% Including caseins 42.6 g 14.2% lipids 56.3 g 18.8% carbohydrates 8.6 g 2.9% salts 0.5 g 0.15% agar-agar 2.0 g 0.67% water 188.1 g 62.7% water (fat-free basis) 188.1 g 77.2% total 300.0 g 100.0%
Example 6: Testing of the Impact of a Gelling Agent on a Fresh Cheese Substitute Preparation
Mixing Calcium Caseinate, Agar-agar, Water, Deodorized Coconut Oil and Glucose:
[0399] 18 g of calcium caseinate casein (Armor Protéines, containing 92% caseins as caseinates; 1% lipids, and 1% calcium), were resuspended in 195 g of water. resulting in a 7.8% (w/w) casein in water suspension. Fifteen grammes of this casein composition were mixed with 2 g of deodorized coconut oil (BioPlanète, France) and 2.5 g of a 25% (w/w) glucose solution in water. The new composition was heated at 45° C., and 4 ml of a heated 0%, 2%, 3% or 4% agar-agar preparation (agar-agar melted in water) was added while gently mixing. Sample 1-4 were duplicated, as samples 5-8 (and samples 1, 4 and 8 were duplicated in a second, independent experiment). The resulting compositions (LpCC) are described in Table 3. pH is about 7.0.
TABLE-US-00004 Composition of the LpCC 1 2 3 4 5 6 7 8 Total weight of LpCC 23.5 g 23.5 g 23.5 g 23.5 g 23.5 g 23.5 g 23.5 g 2 3.5 g casein (w/w) 5.0% 5.0% 5.0% 5.0% 5.0% 5.0% 5.0% 5.0% lipids (w/w) 8.6% 8.6% 8.6% 8.6% 8.6% 8.6% 8.6% 8.6% glucose (w/w) 2.7% 2.7% 2.7% 2.7% 2.7% 2.7% 2.7% 2.7% calcium (w/w) 0.05% 0.05% 0.05% 0.05% 0.05% 0.05% 0.05% 0.05% agar-agar (w/w) - 0.34% 0.51% 0.68% - 0.34% 0.51% 0.68% water (w/w) 83.8% 83.4% 83.2% 83.1% 83.8% 83.4% 83.2% 83.1% Total (w/w) 100.0% 100.0% 100.0% 100.0% 100.0% 100.0% 100.0% 100.0% water (fat-free basis) 91.6% 91.2% 91.0% 90.9% 91.6% 91.2% 91.0% 90.9%
Curdling
[0400] 0.2 g lactic ferments (Streptococcus thermophilus, Lactobacillus bulgaricus, Alsa) were added in samples 1 to 4 (at an initial temperature of 25° C.) but not in samples 5-8 (see Table 4) and the compositions were incubated at 40° C. for 10 hours. In samples 1-4, pH dropped to about 4.5, indicating an active lactic fermentation. In samples 5-8, pH remained stably around 7.0.
Draining
[0401] The entire composition was gently stirred, to disrupt the smooth structure obtained at the highest agar-agar concentrations and placed on a cheesecloth for draining at 4° C. for 16 hours. Curd weights were ranging from about 7 g to about 15 g as described in Table 4. Compositions were estimated (Table 4), based on the assumption that caseins, lipids and agar-agar were entirely retained in the curd. Indeed, little coloration was observed in the drained liquid for samples 1-4, and no coconut oil or agar-agar concretion were observed in the drained liquid at 4° C. In contrast, calcium and glucose estimates are maximal estimates, for part of these compounds may be found in the drained liquid. For the same reason, water content estimates (on a total basis and on a fat-free basis) are minimal estimates.
TABLE-US-00005 Compositions of curds 1 2 3 4* 5 6 7 8 Lactic ferment + + + + - - - Curd weight 7 g* 7 g 9 g 15 g* 2 g 7 g 7 g 12 g* casein 16.7% 16.7% 13.0% 7.8% ND ND ND ND (w/w) lipids (w/w) 28.8% 28.8% 22.4% 13.4% ND ND ND ND glucose (w/w) 8.9% 8.9% 6.9% 4.2% ND ND ND ND calcium (w/w) 0.18% 0.18% 0.14% 0.08% ND ND ND ND agar-agar (w/w) 1.1% 1.3% 1.1% ND ND ND ND water (w/w) 45.5% 44.3% 56.3% 73.5% ND ND ND ND Total (w/w) 100.0% 100.0% 100.0% 100.0% ND ND ND ND water (fat-free basis) 63.8% 62.2% 72.5% 84.9% ND ND ND ND * average of two experiments
[0402] The results show that the gelling agent can be used to modulate moisture, and therefore, the overall composition. With 0.68% agar-agar, more than 60% of the LpCC was retained (vs. 30% without gelling agent), and the product had the casein content and moisture of a fresh cheese, and a plastic texture and aspect, as expected (
[0403] In the absence of lactic fermentation, samples 5-8 gave solid phases of about various sizes, depending an agar-agar concentration. The coloration of the drained liquid suggested that part of casein was lost from the solid phase, and estimates are therefore not indicated in Table 4. The solid phase obtained without ferment were smaller, as compared with its fermented curd counterpart (Table 4), but also kept a loose structure (
Case of the Addition of a Gelling Agent After Curdling
[0404] The same effect could be achieved by adding the gelling agent after curdling: samples: LpCC with a similar composition was incubated for 10 h at 40° C. with lactic bacteria. pH dropped from about 7.0 to about 4.5. After curdling, 3 ml of agar-agar 4% in water was added, as described above, and the total mixture, including curd and liquid phase, was gently stirred, and placed on a cheesecloth for draining.
[0405] Compositions before curdling (LpCC), after curdling and agar-agar addition, and estimates after draining are indicated in Table 5, with the same provisions as above regarding composition after curdling and draining. After draining at 4° C. for 16 hours, curds of about 12 g were obtained in two independent samples, showing a water retention (53% of total) superior to what is observed in the absence of gelling agent (see above).
[0406] The product had the casein content and moisture of a fresh cheese (Table 5), and a plastic texture and aspect, as expected (
[0407] The procedure was replicated in the absence of ferment. A solid phase of lower weight (about 6 g, 27% of total) was obtained. However, texture was very loose, as shown on
TABLE-US-00006 Composition in process for fresh cheese substitute production LpCC After curdling and agar-agar addition Final after draining weight (g) 19.5 g 22.5 g 12 g casein (w/w) 5.0% 4.3% 7.5% lipids (w/w) 10.3% 8.9% 15.4% glucose (w/w) 2.6% 2.2% 3.8% calcium (w/w) 0.05% 0.05% 0.08% agar-agar (w/w) - 0.53% 0.92% water (w/w) 82.1% 83.9% 72.3% Total (w/w) 100% 100% 100.0% water (fat-free basis) 91.5% 92.2% 85.5%
Example 7: Production of Recombinant Alpha and Beta Casein Batch
[0408] The production of recombinant caseins has been described before by others (see description).
[0409] Natural casein genes code for a precursor protein, which includes a signal peptide. In mammals, this peptide is cleaved during casein processing, and is not present in the mature protein in milk. Synthetic genes coding for alpha-S1, and beta casein (related to natural genes P02662 and P02666, respectively), were modified, to remove the signal peptide, and the sequence of the new synthetic open reading frames are shown in Table 6, last column).
TABLE-US-00007 Sequences of natural caseins (precursors) and of the related recombinant proteins. In natural caseins, signal peptides are indicated in bold Name and Uniprot reference of protein precursor sequence of protein precursor sequence of recombinant proteins Alpha-S1 casein P02662 MKLLILTCLVAVALARPKHPIKHQGLPQEVLNENLLRFFVAPFPEVFGKEKVNELSKDIGSESTEDQAMEDIKQMEAESISSSEEIVPNSVEQKHIQKEDVPSERYLGYLEQLLRLKKYKVPQLEIVPNSAEERLHSMKEGIHAQQKEPMIGVNQELAYFYPELFRQFYQLDAYPSGAWYYVPLGTQYTDAPSFS DIPNPIGSENSEKTTMPLW MRPKHPIKHQGLPQEVLNENLLR FFVAPFPEVFGKEKVNELSKDIG SESTEDQAMEDIKQMEAESISSS EEIVPNSVEQKHIQKEDVPSERY LGYLEQLLRLKKYKVPQLEIVPNS AEERLHSMKEGIHAQQKEPMIGV NQELAYFYPELFRQFYQLDAYPS GAWYYVPLGTQYTDAPSFSDIPN PIGSENSEKTTMPLW (SEQ ID NO: 1) (SEQ ID NO: 2) Beta casein P02666 MKVLILACLVALALARELEELNVPGEIVESLSSSEESITRINKKIEKFQSEEQQQTEDELQDKIHPFAQTQSLVYPFPGPIPNSLPQNIPPLTQTPVVVPPFLQPEVMGV SKVKEAMAPKHKEMPFPKYPV EPFTESQSLTLTDVENLHLPLP LLQSWMHQPHQPLPPTVMFPP QSVLSLSQSKVLPVPQKAVPY PQRDMPIQAFLLYQEPVLGPV RGPFPIIV MRELEELNVPGEIVESLSSSEESI TRINKKIEKFQSEEQQQTEDELQ DKIHPFAQTQSLVYPFPGPIPNSL PQNIPPLTQTPVVVPPFLQPEVM GVSKVKEAMAPKHKEMPFPKYP VEPFTESQSLTLTDVENLHLPLPL LQSWMHQPHQPLPPTVMFPPQ SVLSLSQSKVLPVPQKAVPYPQR DMPIQAFLLYQEPVLGPVRGPFP IIV (SEQ ID NO: 3) (SEQ ID NO: 4)
[0410] The resulting ORFs were overexpressed in bacteria, using classical molecular biology technique, and caseins were purified. Eventually, three batches of animal-free recombinant caseins were obtained as calcium caseinates. To obtain concentrated calcium caseinates, casein extracts precipitated with H.sub.2SO.sub.4 at pH=4.6 were resuspended in water, pH was slowly adjusted to 7, with Ca(OH).sub.2, and the preparation was concentrated by evaporation using a Rotavapor R-300 (Buchi)] device. [0411] Batch 1: beta casein (calcium caseinate), 150 mg/g of composition, pH7. [0412] Batch 2: alpha-S1 casein and beta casein (calcium caseinate), about 50 mg/g and 25 mg/g, respectively, pH=7 [0413] Batch 3: alpha-S1 casein and beta casein (calcium caseinate), about 6 mg/g and 3 mg/g, respectively, pH=7
[0414] In these batches, other proteins and other organic molecules were potentially present, but caseins represented more than 90% of total proteins, and were estimated to represent more than ⅔ of dry weight.
Example 8: Testing the Curdling of Partially Purified Recombinant Beta Casein,
[0415] Using batch 1 from example 7, a casein composition containing recombinant beta casein was used to test the curdling capacity of recombinant beta casein, in the absence of any texturing agent. For this, a preparation having approximatively the same casein, lipid, and water content as in sample 1 of example 6 was prepared, but with recombinant beta-casein instead of commercial caseinate.
[0416] Batch 1 preparation was diluted with water to obtain a recombinant beta-casein composition at a concentration of 58.7 mg/g. Twenty grammes of this casein composition was mixed with coconut oil, glucose, and agar-agar melted in water, to a 23.5 g composition containing 5% casein, 8.5% lipids, 2.7% glucose and 0.68% agar-agar in water. Additional carbohydrate and proteins may come from the casein composition as well as salts and are not accounted for in this table. Therefore, water content is only an estimate as well, but given that in the initial casein composition, casein represented at least ⅔ of dry weight, errors in water content should therefore not exceed 3%.
[0417] The composition was seeded with 0.3 g lactic ferments and incubated at 40° C. for 10 hours. pH dropped from about 7.0 to about 4.5. The entire composition was then placed on a cheesecloth for draining at 4° C. for 16 hours. A curd of 7 g could be obtained after draining (
[0418] The ability of alpha-S1 and beta caseins from example 7 to coagulate efficiently at low pH was tested simply by the addition of lactic acid into 90 ml of the low concentration Batch 3 composition, to adjust pH at about 4.5. About 0.7 g of material was obtained after separation by filtration on a cheese cloth (
Example 8: Making Fresh Cheese Substitutes From Partially Purified Recombinant Caseins
[0419] Batch 1 preparation was diluted with water to obtain a recombinant beta-casein composition at a concentration of 78.2 mg/g. Fifteen grammes of this casein composition was mixed with coconut oil, glucose, and agar-agar, as described in example 6, to obtain the composition described in Table 7. Additional carbohydrate and proteins may come from the casein composition as well as salts and are not accounted for in this table. Therefore, water content is only an estimate as well, but given that in the initial casein composition, casein represented at least ⅔ of dry weight, errors in water content should therefore not exceed 3%.
[0420] The composition was seeded with 0.3 g lactic ferments and incubated at 40° C. for 10 hours. pH dropped from about 7.0 to about 4.5. The entire composition was gently stirred and placed on a cheesecloth for draining at 4° C. for 16 hours.
[0421] Curd weight was about 14 g. Its composition (Table 7) was estimated, with the same provisions as above and as in example 6. About 60% of the LpCC was retained. The resulting product had the casein content and moisture desired for fresh cheese. Texture was not as stiff as with commercial calcium caseinate in example 6, but nevertheless appropriate for a fresh cheese, displaying plasticity and firmness to be shaped in stable forms (
[0422] The same procedure was followed, using Batch 2 of casein, containing apha-S1 casein and beta casein. Only 38% of the LpCC was retained, which could be due to the low protein content (2.5% of LpCC), but the resulting product had the casein content and moisture desired for fresh cheese. Texture was not as stiff as with commercial calcium caseinate in example 6, but nevertheless appropriate for a fresh cheese, displaying plasticity and firmness to be shaped in stable forms (
TABLE-US-00008 Composition during process of making an edible composition Nature of casein Beta casein (Batch 1) Alpha-S1 and beta casein (Batch 2) Step LpCC final after curdling and draining LpCC final after curdling and draining Weight (g) 23.5 g 14 g 23.5 g 9 g casein (w/w) 5.0% 8.4% 2.5% 6.4% lipids (w/w) 8.5% 14.3% 8.5% 22.2% glucose (w/w) 2.7% 4.5% 2.7% 6.9% agar-agar (w/w) 0.68% 1.1% 0.68% 1.8% water (w/w) 83.1% 71.7% 85.7% 62.6% Total (w/w) 100.0% 100.0% 100.0% 100.0% water (fat-free basis) 90.9% 83.7% 93.7% 80.5%
Case of the Addition of a Gelling Agent After Curdling
[0423] We also tested the addition of the gelling agent after curdling, following the same procedure as in example 6. Compositions before curdling (LpCC), after curdling and agar-agar addition, and estimates after draining are indicated in Table 8, with the same provisions as above regarding composition after curdling and draining. A curd of about 10 g (44% of total) was obtained after lactic fermentation, addition of agar-agar (0.53%), and draining at 4° C. for 16 hours in a cheesecloth. In the absence of ferment, a solid phase of lower weight (about 6 g, 27% of total) was obtained. However, texture was very loose, as shown on
TABLE-US-00009 Composition during process of making an edible composition LpCC After curdling and agar-agar addition Final after draining weight (g) 19.5 g 22.5 g 10 g casein (w/w) 5.0% 4.3% 9.0% lipids (w/w) 10.3% 8.9% 18.9% glucose (w/w) 2.6% 2.2% 4.7% agar-agar (w/w) - 0.53% 1.13% water (w/w) 82.2% 84.0% 66.3% Total (w/w) 100% 100% 100.0% water (fat-free basis) 91.6% 92.2% 81.7%
Example 10: Making a Soft Cheese Substitute From Partially Purified Recombinant Caseins
[0424] Batch 1 from example 1 was further concentrated, to reach a concentration of 250 mg/g of composition.
[0425] Fifteen grammes sunflower oil, 34 g of hydrated cashew nuts (for the composition of hydrated cashew nuts. see example 5 above), 70 g of water and 1.5 g of agar-agar were mixed. The mixture was brought to the boil and boiled for 10 to 20 seconds. One hundred grammes of the concentrated casein composition, containing 25 g of recombinant beta casein were added to this mixture and gently mixed. LpCC composition is detailed in Table 9.
Curdling. Draining. Salting. and Aging
[0426] 200 g of this LpCC (out of a total of 220.5 g) was seeded with ferments, including lactic bacteria (MBT, SOGEBUL, Dole, France), yeast (DH2d SOGEBUL, Dole, France) and additional ripening ferments (PC12H, SOGEBUL, Dole, France), at a temperature inferior to 35° C. The composition was incubated at 20° C. for 24 hours, in a mold.
[0427] Product was removed from the mold, and set on a grid for draining. The product and grid were placed into a box to control hygrometry. About ¼ to ½ teaspoon of table salt (La Baleine. France), depending on desired taste, can be spread on the upper face and the product is incubated at 14° C. Twenty-four hours later, the product is flipped, and the same salt composition is spread on the other face. The product is incubated at 14° C. on a grid into the refining box to control hygrometry, and flipped every second day, for several weeks. During this period, water is removed from the refining box on a regular manner. The product at day 7 is featured on
[0428] Weight loss consists essentially in water. Since the initial composition is known (with the composition of dry cashew nut being about 22% of carbohydrates. 20% of proteins. and 53% of lipids. and a few percent of water), an evaluation of water loss can be used for the calculation of the product composition, and for achieving the expected composition or a soft cheese, notably in terms of moisture (between 62% and 80% of moisture. on a fat free basis). By day 7, product was estimated to be 150 g, protein and lipid content to be 17.4% (including 15.1% casein and derived peptides) and 16.6%, respectively. and moisture to be 76%. on a fat-free basis.
TABLE-US-00010 Composition of LpCC weight (g) 220.5 g proteins (w/w) 13.1% Including caseins 11.3% lipids (w/w) 12.5% glucose (w/w) 1.2% agar-agar (w/w) 0.68% water (w/w) 72.5% Total (w/w) 100% water (fat-free basis) 82.9%