MICROPARTICULATION OF RECOMBINANT BETA-LACTOGLOBULIN AND ASSOCIATED FOOD APPLICATIONS

Abstract

The present invention relates to a method for the preparation of a microparticulated rBLG (recombinant -lactoglobulin), a microparticulated rBLG obtained from said method and a composition thereof, and the use of such microparticulated rBLG for making animal and non-animal dairy products.

Claims

1. A method for the preparation of microparticulated recombinant beta-lactoglobulin (rBLG) comprising the steps: a) Providing an aqueous solution of rBLG, said aqueous solution comprising from 3 to 20% w/w of rBLG and having a pH between 3 and 8 or between 4 and 8, b) Heating the aqueous solution of rBLG at a temperature comprised between 75 and 99 C., and c) Applying a high shear rate treatment of at least 70 s.sup.1 or at least 100 s.sup.1, to the aqueous solution of rBLG to afford a microparticulated rBLG aqueous solution, the percentage being expressed in weight in relation to the total weight of the aqueous solution.

2. The method according to claim 1, wherein the temperature of the heating step in step b is comprised between 8 and 99 C. or between 82 and 98 C.

3. The method according to claim 1, wherein the rBLG concentration in the aqueous solution of step a is comprised between 5 and 20% w/w or between 8 and 20% w/w, the percentage being expressed in weight in relation to the total weight of the aqueous solution.

4. The method according to claim 1, wherein the pH of the aqueous solution of step a is comprised between 4.5 and 7 or between 5.5 and 6.5.

5. The method according to claim 1, wherein the heating step in step b and the high shear treatment in step c are performed simultaneously.

6. The method according to claim 1, wherein the high shear treatment in step c is performed after the heating step in step b.

7. The method according to claim 1, wherein the method further comprises one or more steps selected among: a step d of cooling the aqueous solution to a temperature lower than 65 C., a step e of concentrating the aqueous solution, and a step f of drying the aqueous solution.

8. The method according to claim 1, wherein the aqueous solution of step a further comprises at least one polysaccharide.

9. The method according to claim 8, wherein the polysaccharide/rBLG ratio is comprised between 1/3 and 1/30.

10. The method according to claim 1, wherein the rBLG is obtained from fungi.

11. A microparticulated rBLG obtained from the process according to claim 1.

12. The microparticulated rBLG according to claim 11, wherein the rBLG is in the form of aggregates, said aggregates having an average particle size comprised between 0.1 and 15 m, between 1 and 10 m, or between 1 and 6 m.

13. A dry composition comprising rBLG comprising 45-95% w/w of microparticulated rBLG and at least one polysaccharide, the percentage being expressed in weight in relation to the total weight of the dry composition.

14. The dry composition according to claim 12, wherein the microparticulated rBLG is in the form of aggregates, said aggregates having an average particle size comprised between 0.1 and 15 m, between 1 and 10 m, or between 1 and 6 m.

15. An aqueous solution of microparticulated rBLG, wherein the solution comprises from 2 to 20% w/w of microparticulated rBLG, 0.1 to 5% w/w of polysaccharide and water, the percentage being expressed in weight in relation to the total weight of the aqueous solution.

16. (canceled)

17. The method according to claim 10, wherein the fungi is of the genus Aspergillus.

18. An animal or non-animal dairy product made using the microparticulated rBLG according to claim 11.

19. An animal or non-animal dairy product made using the dry microparticulated rBLG composition according to claim 13.

20. An animal or non-animal dairy product made using the aqueous solution of microparticulated rBLG according to claim 15.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0345] FIG. 1 represents the average diameter d50.3 of the particle size distribution of microparticulated rBLG produced in different conditions of rBLG concentrations, temperature and pH of the aqueous solution.

[0346] FIG. 2 represents the cumulative frequency of the particle size distribution (average of 3 replicates) of the microparticulated rBLG sample produced at 90 C., pH 6.0, 10% w/w protein and 500 s.sup.1 shear rate. A second curve has been obtained of the microparticulated sample after heat stability test (90 C./10 min).

[0347] FIG. 3 is a scanning electron microscope (SEM) picture of microparticulated rBLG.

[0348] FIG. 4 is pictures showing yoghurts H1, H2, H3 and S1, S2, S3 that contain the ingredients reported in table 12 and that have the proximate composition reported in table 13

[0349] FIG. 5 represents the decrease of pH over time for solutions prepared from microparticulated rBLG at a concentration of 9% w/w and microparticulated whey protein at a concentration of 9% w/w after addition of 2% w/w glucono delta lactone (GDL).

[0350] FIG. 6 represents the lipase activity in microparticulated rBLG and rBLG solutions.

EXAMPLES

[0351] The present invention is further illustrated by the following examples.

Example 1: Production of Microparticulated rBLG at a Lab Scale

[0352] rBLG used in the example 1 was produced by precision fermentation and has the composition as defined in table 1.

TABLE-US-00001 TABLE 1 rBLG composition in example 1. Total carbohydrates correspond to polysaccharides. Composition Content Unit Total proteins (N % 6.25) 76.2 g/100 g powder Beta lactoglobulin 75 g/100 g powder Total fat 0.6 g/100 g powder Total carbohydrates 19.2 g/100 g powder Moisture 3.0 g/100 g powder Ash 1.6 g/100 g powder Cu 2.38 (+0.71) mg/100 g powder Ca 413.0 (+123.9) mg/100 g powder Fe 4.04 (+1.21) mg/100 g powder Mg 110.0 (+33.0) mg/100 g powder P 30.8 (+9.2) mg/100 g powder K 54.3 (+16.3) mg/100 g powder

General Procedure

[0353] Solutions of rBLG were prepared by rehydrating rBLG powder in demineralized water. pH of the solutions was adjusted to pH 4 to 6 with HCl 1M. The solutions were covered to avoid evaporation of water, and heated at a temperature of 70, 80 and 90 C. for 20 minutes with simultaneous stirring at a shear rate of 500 s.sup.1. The solutions were finally cooled to 6 C. without stirring.

[0354] Simultaneous heat treatment and high shear was obtained using a rotational rheometer MCR 502 (Anton Paar Germany GmbH, Ostfildern, Germany) equipped with a coaxial cylinder geometry.

Tests

[0355] The solutions after simultaneous heat and shear treatments and cooling were subjected to various tests: particle size distribution, heat stability and acid stability.

Particle Size Distribution

[0356] The volume-weighted particle size distributions of protein solutions after heating, shearing and cooling were determined by static light scattering (Beckman Coulter LS 13 320 fitted with a Universal Liquid Module and control software v6.01, Beckman Coulter Inc., Miami, FL, USA). Between 100 and 300 L of sample was added to the measurement chamber. An obscuration of between 3 and 7% with maximum Polarization Intensity Differential Scattering (PIDS) of 60% was adhered for all particle size measurements. All measurements were performed at room temperature (18-20 C.) and the preparations were run in threefold and in three measurement cycles for each measurement. A refractive index of 1.52 was used for BLG (designation: 152.rf780d PIDS included). The refractive index of the solvent (water) was 1.33. An imaginary refractive index of 0.00 was used for the particles and the solvent (LS 13 320 Particle Size Analyzer Manual. Instruction for Use LS 13 320 Laser Diffraction Particle Size Analyzer; Beckman Coulter: Brea, CA, USA, 2011).

Heat Stability Test

[0357] After simultaneous heat treatment and high shear rate treatment and cooling, protein solutions were poured in 2 ml tubes and placed in a 90 C. water batch during 10 min, then quickly cooled down using ice bath. Solutions were observed where absence or presence of a gel, a flocculate or a precipitate is noted.

[0358] In addition to visual observations, heat stability of the microparticulated protein solutions were also evidenced by comparing particle size distribution before and after the heat stability test (90 C., 10 min).

Acid Stability Test

[0359] GDL (Glucono-Delta Lactone) (0.75% w/w) was added to the solutions of protein after being submitted to simultaneous heat treatment and high shear rate treatment. The solutions were then left unstirred, in a steam room at 27 C., until pH 4.50 is reached, then stored at 5 C. during 24 h. After 24 hours, solution was observed to note if a gel was formed or not.

Results

[0360] Results are presented below in tables 2 and 3.

TABLE-US-00002 TABLE 2 Impact of heating temperature, pH and protein concentration under high shear conditions (500 s.sup.1), on particle size (m). The d10.3, d50.3 and d90.3, represent the particle size distribution, i.e. the particle sizes at which 10, 50 and 90% of the particle volume is below or equal. The d3.2 is a mean diameter of the size distribution also called the sauter diameter. rBLG concentration Temperature (% w/w) pH ( C.) d90.3 d50.3 d10.3 d3.2 2.5 4.5 70 16.44 8.10 3.96 7.06 2.5 4.5 80 23.29 8.35 2.71 5.93 2.5 6 80 15.92 6.52 2.94 5.56 2.5 4.5 90 21.61 11.28 5.60 9.53 2.5 6 90 16.67 6.32 1.84 3.71 5 4.5 80 8.82 4.80 2.83 4.50 5 6 80 10.17 5.67 3.04 5.00 5 4.5 90 9.16 5.47 3.06 4.90 5 6 90 11.31 6.00 3.06 5.27 10 4.5 80 7.79 3.69 2.41 3.68 10 6 80 8.09 4.42 2.85 4.34 10 4.5 90 6.92 3.75 2.83 3.92 10 6 90 8.84 5.03 2.89 4.63

[0361] Particle sizes d50.3 between 11.28 and 3.69 m were achieved and can be considered as a suitable range of microparticle sizes to improve creaminess in dairy applications. At a concentration of 2.5% larger particles were achieved at pH 4.5, while at higher concentrations (5%, 10%) slightly larger particles were obtained at pH 6.0 rather than 4.5. There was also observed a decrease in particle size with increasing concentration of protein (FIG. 1).

[0362] Samples comprising microparticulated rBLG and obtained according to the process described above, were submitted to heat stability test (as described above).

[0363] Results are reported in table 3 (visual analysis) and in FIG. 2 (comparison of particle size distribution before and after heat stability test). Microparticulated rBLG samples produced at 90 C. (pH 4.5 or 6.0) from 2.5, 5.0 and 10% protein concentration under 500 s.sup.1 shear rate, which already gave suitable size of microparticules (table 2) are also stable: the samples are homogeneous, do not show phase separation, sedimentation or flocculation. As a confirmation, no significant differences between particles size distributions obtained before and after heat stability test (90 C./10 min) were reported on the samples that were visually considered as stable. The example of the microparticulated rBLG sample produced at 90 C., pH 6.0, 10% w/w protein and 500 s.sup.1 shear rate, is reported in FIG. 2. Particle size is unchanged after heat stability test, which further evidences heat stability in these conditions.

TABLE-US-00003 TABLE 3 Heat stability study performed on rBLG solutions microparticulated under high shear (500 s.sup.1) at 90 C. and pH 4.5 and 6.0. Protein concentration pH pH Conditions Temperature (%) 4.5 6.0 A 90 C. 2.5 stable stable B 90 C. 5 stable stable C 90 C. 10 stable stable

[0364] Samples comprising microparticulated rBLG and obtained according to the process described above in conditions C described in Table 3 (90 C.; pH 6.0 under high shear (500 s.sup.1) from a 10% w/w initial rBLG solution, were submitted to acid stability test (as described above). The test resulted in no gel formation and no formation of visible particles/aggregates. The sample was liquid after the test which evidenced the stability of the microparticulated rBLG produced in conditions C against acidification to pH 4.5. On the contrary, microparticulated rBLG produced in conditions A were not stable and visible aggregates were observed. Thus, only the microparticulated rBLG obtained with the process of the present invention (condition C) are stable enough, even against acidification, which makes suitable for the production of animal and non-animal dairy products.

Example 2: Microparticulated rBLG Obtained Using a Scraped Surface Heat Exchanger (130 ml Batch, Around 10 g Protein/Batch)

General Procedure

[0365] rBLG powder used here was produced by precision fermentation and has the composition as defined in table 1.

[0366] Solutions of 10% w/w of rBLG were prepared by rehydrating rBLG powder in demineralized water. pH of the solutions was adjusted to pH 6 with HCl 1M.

[0367] Microparticulation treatment with laboratory scale Scraped Surface Heat Exchanger (SSHE) (technical workshop of the university of Hohenheim, Stuttgart, Germany) was performed as described by (Filla et al., 2021; Protte et al., 2017): 130 mL of unheated rBLG solution was poured into the device. First, shear treatment was started by increasing stirrer to 200 rpm (corresponding to a representative shear rate of 65 s.sup.1) or 1000 rpm (corresponding to a representative shear rate of 327 s.sup.1), followed by initiation of the heat treatment. Solutions were heated via a water bath at 95 C. (temperature of the solution was 90 C.) connected to the double jacket of the SSHE for 20 min. After the heat treatment, the water supply was switched to cooling water (10 C.) for 10 min. Samples were stored cooled at 6 C.

Tests

[0368] The solutions after simultaneous heat and shear treatment and cooling were subjected to various tests: particle size distribution, heat stability and acid stability as described in example 1.

Results

[0369] Particle size of microparticulated rBLG were measured according to the methology described in Example 1. Results are presented in table 4 below.

TABLE-US-00004 TABLE 4 Particle size of microparticulated rBLG produced under high shear treatment (200 or 1000 rpm, correponding respectively to 65 s.sup.1 and 327 s.sup.1) at 90 C., pH 6 from 5 and 10% w/w rBLG aqueous solution. rBLG High concetration shear d50.3 Condition (%) (rpm) (m) K 5 1000 3.15 (invention) L 10 200 26.65 (comparative) M 10 1000 2.63 (invention)

[0370] Treatment of rBLG aqueous solution (5 and 10% w/w) at 90 C. and pH 6.0 under high shear at 327 s.sup.1 (i.e. 1000 rpm) delivered microparticulated rBLG with a suitable size of d50.3 lower than 15 m (Table 10). At a lower shear rate (65 s.sup.1), particle size was much bigger, with a diameter d50.3 of 26.65 m.

[0371] Heat stability of microparticulated rBLG was also evaluated according to the methodology of example 1.

[0372] Microparticuated rBLG solutions obtained under conditions presented in Table 4 (conditions L and M) were submitted to heat stability test. Particle size before and after the test was measured using methodology of example 1.

[0373] Results are summarized in table 5.

TABLE-US-00005 TABLE 5 Particle size of microparticulated rBLG (d50.3 in m) subjected to heat stability test or freeze-drying. d50.3 d50.3 d50.3 after after heat after High micro- stability freeze- shear particulation test drying Conditions (rpm) (m) (m) (m) L 200 26.65 26.2 (comparative) M 1000 2.63 2.6 2.71 (invention)

[0374] Heat stability test showed that there was no change of the particle size before and after the heat stability test (90 C. for 10 min), demonstrating heat stability of microparticuled rBLG (table 5).

[0375] In addition to the heat stability, stability of microparticulated rBLG to freeze drying was evaluated. Microparticulated rBLG solutions (condition M, table 4) were freezed at 40 C. in small metal dishes. Frozen samples in dishes were placed in freeze dryer with 40 C. for 3 days. Powder was filled separately in a bag and vacuum packaged. Particle size of freeze dried microparticulated rBLG was measured and compared to particle size before freeze drying. No alteration of the particle size was observed after freeze drying (table 5), which demonstrates stability of microparticulated rBLG to freeze-drying.

Example 3: Morphology and Size of Microparticulated rBLG

[0376] A 2% w/w aqueous solution of microparticulated rBLG was prepared by rehydrating at room temperature in demineralized water microparticulated rBLG powder obtained with the methodology presented in Example 2 using conditions M (i.e. initial rBLG concentration was 10% w/w, shear rate was 327 s.sup.1 using laboratory scale SSHE, pH 6.0 and temperature at 90 C.) followed by freeze-drying. This solution was analyzed by scanning electron microscopy (SEM).

[0377] 5 l of 2% w/w microparticulated rBLG solution was deposited on a sillicon wafer. The drop was spread using a cone to ensure good distribution and it was let to dry. Afterwards the sample was covered with copper metallization. Images were collected by using a SEM FEI quanta 250 FEG (Fiel Emission Gun) equipment.

[0378] Pictures of microparticulated rBLG are presented in FIG. 3. The sample is composed of spherical aggregates ranging from 1 to 10 m (FIG. 3).

Example 4: High Protein Yogurts Comprising Microparticulated rBLG

[0379] Yogurts have been prepared from microparticulated rBLG and/or rBLG in presence or absence of other protein sources (basic formula or hybrid formula) or with starch (potato starch ETENIA 457) and properties of yogurts have been analyzed.

General Procedure

[0380] rBLG used in the Example 4 was produced by precision fermentation and has the composition as defined in table 6.

TABLE-US-00006 TABLE 6 rBLG composition in example 6. Total carbohydrates correspond to polysaccharides. Composition Content Unit Total proteins (N 6.25) 79.7 g/100 g powder Beta lactoglobulin 79.7 g/100 g powder Total fat 0.6 g/100 g powder Total carbohydrates 13.9 g/100 g powder Moisture 3.0 g/100 g powder Ash 3.5 g/100 g powder Cu 1.82 (+0.55) mg/100 g powder Ca 144.0 (+43.2) mg/100 g powder Fe 3.25 (+0.98) mg/100 g powder Mg 78.5 (+23.6) mg/100 g powder P 266.0 (+79.8) mg/100 g powder K 1180.0 (+354) mg/100 g powder

[0381] Microparticulated rBLG used in the example 4 has been obtained using the procedure described in example 2 with conditions M (i.e. initial rBLG concentration was 10% w/w, shear rate was 327 s.sup.1 using laboratory scale SSHE, pH 6.0 and temperature at 90 C.) followed by freeze-drying.

[0382] Model yoghurts of example 4 were produced using the following ingredients: Demineralized water; Organic coconut milk (KARA); Glucono-Delta-lactone (GDL); Pasteurized Whole Milk; Potato starch (ETENIA 457, Avebe).

[0383] The model yoghurts were produced using the following procedure: mixing ingredients with demineralized water under moderate agitation at room temperature in a beaker; pre-heating at 40 C. under moderate agitation for 10 min using a water bath; homogenizing the mix for 1 min at 13 500 rpm using an Ultraturrax; pasteurizing the mix at 85 C. for 10 min under moderate agitation using a water bath; cooling the mix to 27 C. by placing the beaker in a cooling cell; adding appropriate amount of GDL in powder form to the mix (between 1 and 2%) and leaving the mix unstirred at 27 C. during 3 hours in order to reach pH 4.5 in aseptic 40 ml polypropylene container; cooling the acidified mix at 4 C. in refrigerator overnight.

[0384] Yogurts compositions (before or after addition of GDL) are summarized in tables 7 and 8.

TABLE-US-00007 TABLE 7 Compositions of yogurts comprising rBLG and microparticulated rBLG (mBLG) in % w/w in relation to the total weight of the composition. H1 and S1 do not comprise rBLG or microparticulated rBLG. Whole Coco Potato Water milk milk starch rBLG mBLG F1 68.53 27.78 3.69 3% rBLG (comparative) F2 61.15 27.78 11.07 9% rBLG (comparative) F3 60.08 27.78 3.69 8.45 3% rBLG 6% mBLG (invention) H1 100 (control) H2 92.62 7.38 6% rBLG (comparative) H3 91.55 8.45 6% mBLG (invention) S1 67.8 27.8 4.4 (control) S2 56.8 27.8 4.4 11.07 9% rBLG (comparative) S3 55.1 27.0 4.4 12.67 9% mBLG (invention)

TABLE-US-00008 TABLE 8 Proximate composition of yoghurts (in g/100 g product) of example 4 comprising rBLG and microparticulated rBLG (mBLG). Proteins Proteins Proteins Total Total from from from Total g/100 g solids Protein rBLG mBLG Milk Lipids Carbohydrates F1 9.1 3.5 3 4.4 0.5 3% rBLG (comparative) F2 16.3 9.5 9.0 4.4 1.5 9% rBLG (comparative) F3 17.3 9.5 3.0 6.0 4.4 2.1 3% rBLG 6% mBLG (invention) H1 12.0 3.3 3.3 3.3 3.5 (control) H2 18.3 9.1 6.0 3.1 3.1 4.3 6% rBLG (comparative) H3 19.2 9.0 6.0 3.0 3.0 4.8 6% mBLG (invention) S1 9.6 0.5 4.4 4.0 (control) S2 20.1 9.5 9.0 4.4 5.4 9% rBLG (comparative) S3 21.7 9.5 9.0 4.4 6.3 9% mBLG (invention)

Tests

Texture Analysis 20 mL yoghurts are produced in aseptic 40 ml polypropylene container for textural analysis using the method outlined below. A TA.XTPlus Texture Analyzer with a 5 kg load cell (Stable Micro System, Surrey UK) was used to characterize the physical properties of the yoghurts.

[0385] Samples were held in refrigerator at 4 C. overnight after production. Prior to testing they were equilibrated for 2 h to room temperature. A cylindrical probe (P/10 10 mm diameter) was used to perform texturometry tests at room temperature (20 C.) (One-Cycle Compression Test). The initial parameters were: pre-test speed, 1.00 mm/s; test speed, 5.00 mm/s; post-test speed, 5.00 mm/s; compression distance, 10.00 mm; trigger force, 5.00 g. The results included hardness (in g, defined as the maximum peak force during the compression), and consistency (or work of penetration in g.Math.sec, defined as the area under the peak corresponding to the compression) are reported in table 9. The tests were carried out in duplicate.

Rheology

[0386] Viscosity of the yoghurts was determined at 10 C. using a Anton Paar MCR95 rheometer equipped with plan-plan geometry (parallel plate fixture with a gap of 1 mm between the fixture (PP25, 25 mm diameter) and sample plate was used) over a range of shear rates (from 0.01 to 100 s1). Viscosity readings were plotted versus shear rate and viscosity at 50 s.sup.1 shear rate was reported in table 9.

[0387] Texture (consistency and hardness), viscosity, coagulation after pasteurization, olfactive properties of yogurts were analyzed, and results are reported in table 9.

TABLE-US-00009 TABLE 9 Measured properties of yogurts obtained from compositions described in table 9. Pasteurization was performed at 85 C. for 10 min; Coagulation was determined with H1 as reference; Smell was compared to odour of H1, and was noted +when an unpleasant odour was observed and when no odour was detected compared to H1; nd = not determined. Coagulation Consis- Hard- Viscosity (Pa .Math. s) Com- after tency ness (at 50 s.sup.1 shear position pasteurization (g. sec..sup.1) (g) rate and 10 C.) Smell F1 NO 71 49.2 4.1 + F2 YES 304 242 298.3 ++ F3 NO 64 49.9 5.4 + H1 NO 19 11 0.62 0 H2 YES 607 440 29.7 + H3 NO 26 14.9 1.8 S1 NO 26 17 5.9 nd S2 YES 55 35 45.6 nd S3 NO 31 22.1 10.6 nd

[0388] As reported in table 9, among all yogurt mixes, the ones that contain rBLG above 6% (F2, H2 and S2) have shown thermal coagulation during pasteurization treatment. In contrast, mixes that contain a low content of rBLG (F1, 3% w/w), no added proteins (H1 and S1) and microparticulated rBLG even in high concentration (F3, H3, and S3) (up to 9% w/w) did not show coagulation during pasteurization. The protein enrichment of yogurt with rBLG (at least from 6% w/w enrichment) in order to reach high protein content (9% w/w total proteins in example 6) and hence produce high protein products led to premature thermal coagulation of the mix during pasteurization, as opposed to protein enrichment with microparticulated rBLG, which did not induce premature thermal coagulation of the mix during pasteurization even when added in high concentration (up to 9% w/w).

[0389] Results of texture of finish yogurts (hardness and consistency) presented in table 9 showed that the addition in the mix of microparticulated rBLG to reach high protein content (F3, H3, S3) either did not increase or increased slightly hardness and consistency vs. control with low or no protein content. For example, F3 with 6% w/w microparticulated rBLG on top of 3% w/w rBLG (total protein content at 9% w/w) showed a hardness of 49.9 g vs. F1 that contains only 3% rBLG which showed a hardness of 49.2 g. For example, H3 with 6% w/w microparticulated rBLG on top of 3% w/w milk proteins showed a hardness of 14.9 g vs. H1 that contains only 3% w/w milk proteins which showed a hardness of 11 g. For example, S3 with 9% w/w microparticulated rBLG in starch based formula showed a hardness of 22.1 g vs. H1 that contains only 3% rBLG which showed a hardness of 17 g. Similar results were obtained on consistency. Similar results were obtained on viscosity. In contrast, addition in the mix of rBLG to reach high protein content (F3, H3, S3) did increase drastically hardness, consistency and viscosity vs. control yogurts with low or no protein content or vs. high protein yogurts at the same total protein content (i.e 9% w/w) but obtained with the addition of microparticulated rBLG. For example, F2 with 6% w/w rBLG on top of 3% w/w rBLG (total protein content at 9% w/w) showed a viscosity of 298.3 Pa.Math.s which is much higher than F1 (4.1 Pa.Math.s) and F3 (5.4 Pa.Math.s) that contains respectively only 3% rBLG (F1) and 6% of microparticulated rBLG on top of 3% rBLG (F3). For example, H2 with 6% w/w rBLG on top of 3% w/w milk proteins (total protein content at 9% w/w) showed a viscosity of 29.7 Pa.Math.s which is much higher than H1 (0.62 Pa.Math.s) and H3 (1.8 Pa.Math.s) that contains respectively only 3% milk proteins (H1) and 6% of microparticulated rBLG on top of 3% milk proteins (H3). For example, S2 with 9% w/w rBLG in starch based formula showed a viscosity of 45.6 Pa.Math.s which is much higher than S1 (5.9 Pa.Math.s) and S3 (10.6 Pa.Math.s) that contains respectively no protein (S1) and 9% of microparticulated (S3). Similar results were obtained on hardness and consistency (table 9).

[0390] The protein enrichment of yogurt with rBLG (at least from 6% w/w enrichment) in order to reach high protein content (9% w/w total proteins in example 6) and hence produce high protein products led to inappropriate texture showing too high viscosity, hardness or consistency, as opposed to protein enrichment with microparticulated rBLG, which only induced slight changes in texture even when added in high concentration (up to 9% w/w).

[0391] The previous results are confirmed by the yogurt pictures presented in FIG. 4 (yogurts H1, H2, H3 and S1, S2, S3). Both H1 containing only 3% milk protein and H3 that contains 6% of microparticulated rBLG on top of 3% milk proteins (total protein content at 9% w/w) showed typical yogurt aspect, H3 having a slightly more compact and smooth aspect than H1. In contrast, H2 yogurts that contains 6% w/w rBLG on top of 3% w/w milk proteins (total protein content at 9% w/w) showed more heterogenous and more solid aspect, closer to heat-induced gel of whey proteins. Regarding the aspects of starch based yogurts (S1, S2 and S3), both S1 containing no protein and S3 that contains 9% of microparticulated rBLG showed very smooth, shiny and typical stirred yogurt aspect. In contrast, S2 yogurts that contains 9% w/w rBLG showed more heterogenous, closer to heat-coagulated whey protein gel.

Example 5: Foaming Properties of Microparticulated rBLG

General Procedure

[0392] A 10% w/w aqueous solution of microparticulated rBLG was prepared by rehydrating at room temperature in demineralized water microparticulated rBLG powder obtained with the methodology presented in Example 2 using conditions M (i.e. initial rBLG concentration was 10% w/w, shear rate was 327 s.sup.1 using laboratory scale SSHE, pH 6.0 and temperature at 90 C.) followed by freeze-drying.

[0393] Another 10% w/w aqueous solution of microparticulated whey protein was prepared by rehydrating at room temperature in demineralized water microparticulated whey protein powder (WPC 550, NZMP).

[0394] 50 mL glass graduated cylinders were filled with 18 mL of solutions and then submitted to intense agitation with plunging Ultraturrax operated at 20 500 rpm for 1 min allowing air incorporation in the solutions. Difference in volume of solutions before and after agitation indicates the quantity of air incorporated in the solution and relates to the foaming capacity of the protein.

Results

TABLE-US-00010 TABLE 10 Volume (in mL) of microparticulated solutions of protein (microparticulated rBLG versus microparticulated whey protein) (10% w/w protein) before and after intense agitation treatment (Ultraturrax at 20500 rpm during 1 min). Before stirring After stirring Microparticulated Whey proteins 18 33 Microparticulated rBLG 18 21

[0395] Comparison of the volume a 10% w/w of microparticulated rBLG solution before and after stirring with Ultraturrax at 20500 rpm shows a small increase. Before stirring the volume was 18 mL, whereas after stirring a volume of 21 mL was measured (Table 10). On the contrary, the same experiment carried out on 10% w/w microparticulated whey protein solution showed that the volume of the solution increased from 18 mL to 33 mL. The foaming effect of microparticulated rBLG is very low, making it suitable for the preparation of dairy products, as air incorporation in mixes can be detrimental to powder rehydration step and further steps of pumping, heat exchange or highpressure homogenization.

Example 6: Buffering Capacity of Microparticulated rBLG

General Procedure

[0396] A 9% w/w aqueous solution of microparticulated rBLG was prepared by rehydrating at room temperature in demineralized water microparticulated rBLG powder obtained with the methodology presented in Example 2 using conditions M (i.e. initial rBLG concentration was 10% w/w, shear rate was 327 s.sup.1 using laboratory scale SSHE, pH 6.0 and temperature at 90 C.) followed by freeze-drying.

[0397] Another 9% w/w aqueous solution of microparticulated whey protein was prepared by rehydrating at room temperature in demineralized water microparticulated whey protein powder (WPC 550, NZMP).

[0398] Appropriate amount of glucono delta lactone (GDL) in powder form was added to reach 2% w/w GDL in the solutions. pH of the solutions was then monitored as a function of time during acidification (FIG. 5).

Results

[0399] FIG. 5 shows a difference in acidification rate between the two 9% w/w protein solutions. Acidification was much faster in the 9% w/w microparticulated rBLG than in the 9% w/w microparticulated whey protein solution, which means that the buffering effect of microparticulated rBLG solution is lower and results in a faster gelling by acidification. Then, a reduced process time is required for the acidification step, especially when high amount of protein need to be acidified, which is the case in the production of high protein yogurt without straining.

[0400] In addition, the lower buffering capacity of microparticulated rBLG will result in a lower quantity of acid (e.g. lactic acid) needed to be added or produced by lactic cultures to reach target pH (around 4.5) of finish products (for example, yogurts, fresh cheese etc.) which will result in improved organoleptic properties.

Example 7: High Protein UHT (Ultra High Temperature) Milks Comprising Microparticulated rBLG

[0401] UHT milks were prepared from rBLG and microparticulated rBLG solutions.

General Procedure

[0402] Aqueous solutions of microparticulated rBLG at different protein concentrations (from 3% w/w to 9% w/w) were prepared by rehydrating at room temperature in demineralized water microparticulated rBLG powder obtained with the methodology presented in Example 2 using conditions M (i.e. initial rBLG concentration was 10% w/w, shear rate was 327 s.sup.1 using laboratory scale SSHE, pH 6.0 and temperature at 90 C.) followed by freeze-drying.

[0403] Aqueous solutions of rBLG in example 7 at different protein concentrations (from 3% w/w to 9% w/w) were prepared by rehydrating at room temperature in demineralized water rBLG powder obtained by precision fermentation and that has the composition as defined in table 11.

TABLE-US-00011 TABLE 11 rBLG composition in example 7. Total carbohydrates correspond to polysaccharides. Composition Content Unit Total proteins (N 6.25) 81.2 g/100 g powder Beta lactoglobulin 81.2 g/100 g powder Total fat <0.5 g/100 g powder Total carbohydrates 10.4 g/100 g powder Moisture 5.3 g/100 g powder Ash 1.6 g/100 g powder Cu 1.59 mg/100 g powder Ca 11.9 mg/100 g powder Fe 3.8 mg/100 g powder Mg 65.3 mg/100 g powder P 88.9 mg/100 g powder K 601 mg/100 g powder

[0404] Solutions were prepared in batches of 15 ml. pH was adjusted to 6.7 with 1 M NaOH. Approximately 5.5 mL was filled in stainless steel heating tubes. Experiments were performed in duplicates. Batch UHT heating with lab scale UHT (technical workshop of the University of Hohenheim, Stuttgart, Germany): heating tubes were placed in the pressure chamber (hanging vertically). Heating of the samples was rapidly realized by saturated steam condensation and lasted for 30 seconds. Cooling was realized within 30 seconds with ice-water. Heating temperature for these trials was 140 C. (steam 3 bar). Size particle was measured before and after heat treatment to assess the heat stability of the solutions and using the methodology presented in example 1.

[0405] Particle size was determined before and after UHT treatment of rBLG and microparticulated rBLG, and results are reported in tables 12 and 13. UHT treatment consists in heating the solution at 140 C. for 30 seconds with a heating ramp of 10 seconds and a cooling ramp of 30 seconds.

Results

TABLE-US-00012 TABLE 12 Particle size defined by d90.3, d50.3, d10.3 and Sauter diameter (d3.2) of solutions of rBLG before and after UHT treatment at pH 6.7 and 4.5 and as a function of protein concentration in solution. rBLG Sauter UHT concentration d90.3 d50.3 d10.3 diameter pH treatment (% w/w) (m) (m) (m) (m) 6.7 Before 3 2.2 0.3 0.1 0.9 After 3 2.5 0.4 0.2 1.1 After 4 2.8 0.6 0.3 1.1 After 5 3.8 1.0 0.4 2.6 After 7 170.9 6.0 1.0 97.8 After 9 222.9 53.3 4.1 108.4 4.5 After 3 493.1 200.1 18.5 190.0

TABLE-US-00013 TABLE 13 Particle size defined by d90.3, d50.3, d10.3 and Sauter diameter (d3.2) of solutions of microparticulated rBLG (mBLG) before and after UHT treatment at pH 6.7 and 4.5 and as a function of protein concentration in solution. mBLG Sauter UHT Concentration d90.3 d50.3 d10.3 diameter pH treatment (% w/w) (m) (m) (m) (m) 6.7 Before 3 7.6 3.9 2.7 2.0 After 3 5.1 3.1 2.2 1.6 After 4 6.6 3.4 2.4 1.8 After 5 6.5 3.3 2.4 1.7 After 6 6.9 3.6 2.4 2.0 After 7 6.9 3.7 2.3 2.1 After 8 5.3 3.2 2.3 1.6 After 9 5.9 3.3 2.3 1.6 4.5 After 3 5.4 3.3 2.3 1.7 After 6 5.1 3.2 2.2 1.8 After 9 5.1 3.2 2.2 1.5

[0406] At pH 6.7, rBLG is stable to UHT treatment when the concentration is equal or below 4% w/w (tables 12) as evidenced by the particle size that remained unchanged after UHT treatment. Above 5% w/w of rBLG, the solution coagulates (the Sauter diameter increases up to 108.4 m). On the contrary, under the same conditions, microparticulated rBLG is stable to the UHT treatment for microparticulated rBLG solution comprising from 3 to 9% w/w protein as evidenced by the particle size that remains unchanged as compared to before UHT treatment (Table 13). Mixtures of rBLG (3% w/w) and microparticulated rBLG (3 to 6% w/w) are also stable when treated under the UHT conditions (results not shown).

[0407] Under acidic conditions (pH 4.5), a 3% w/w rBLG solution is not stable (Table 12) as shown by the Sauter dimeter of 190 m. This is not the case of a 3% w/w microparticulated rBLG solution. Under the same conditions, the Sauter diameter remains constant around 1 or 2 m for microparticulated rBLG solution of 3 to 9% w/w protein (Table 13) which evidenced UHT stability.

Example 8: Lipase Activity and Microparticulation Process

[0408] Lipase activity was screened in rBLG sample and microparticulated rBLG samples.

[0409] Recombinant BLG ingredients can contain enzymes produced by microbial hosts. Some of these enzymes are degrading lipids in food product applications which generate volatile compounds involved in detrimental off-notes. Enzymes with lipase activity can have similar size and molecular weight than BLG. Therefore, it is difficult to separate them by non-thermal physical process (frontal filtration, tangential filtration or centrifugation).

Procedure

Protein Samples rBLG sample was produced by precision fermentation and has the composition as defined in table 1 of example 1. Microparticulated rBLG was obtained from this rBLG sample with the methodology presented in Example 2 using conditions M (i.e. initial rBLG concentration was 10% w/w, shear rate was 327 s.sup.1 using laboratory scale SSHE, pH 6.0 and temperature at 90 C.) followed by freeze-drying.

Lipase Activity Assay

[0410] Lipase substrate specificity was determined using the pNP-assay (p-nitrophenyl palmitate) according to Stemler and Scherf (2022). Stock solutions (10 mmol/L) of pNP-derivatives were prepared in acetonitrile: 2-propanol (1:4, v:v) and stored frozen. The stock solutions were diluted with an assay buffer (50 mmol/L Tris-HCl, 1 mmol/L CaCl2, 0.3% Triton X-100, pH 7.5) directly before use. Lipases were dissolved at 1 mg/mL in the lipase buffer (50 mmol/L Tris-HCl, 1 mmol/L CaCl2, pH 7.5). For calibration, p-nitrophenol solutions from 0.05 mmol/L to 0.25 mmol/L were prepared using an assay buffer. The analysis was carried out in 96-well plates. Lipase solution (10 L) was added to 190 L of substrate working solution or 190 L of assay buffer (lipase control) or 190 L of calibration solutions (calibration). The absorbance at 410 nm was recorded at 30 C. for 60 min. The absorbance of the released p-nitrophenol was corrected by subtracting both lipase control and substrate control at the corresponding time. Lipase activity values of the samples were normalized to protein concentration of the sample solutions.

Results

[0411] Results of lipase activity for rBLG and Microparticulated rBLG are presented in FIG. 6. A sharp decrease in lipase activity was measured in microparticulated rBLG sample as compared to rBLG sample. This drastic reduction of lipase activity is also correlated with a decrease in unpleasant smell in coconut fat based yogurts formulated with microparticulated rBLG as compared to the ones formulated with rBLG (table 9).

[0412] It was then found that lipase was present in the rBLG samples. However, following microparticulation process according to the invention, lipase activity was decreased. The microparticulation process is thus responsible of the drastic reduction of lipase activity which improves the organoleptic properties of product applications, such as the one that contain lipids.