WO2025088607A1

WO2025088607A1 - Milk protein substitute and products made therefrom

Info

Publication number: WO2025088607A1
Application number: PCT/IL2024/051026
Authority: WO
Inventors: Arie Abo; Lilach ZATTELMAN; Chana SOKOLIK
Original assignee: Imagindairy Ltd
Current assignee: Imagindairy Ltd
Priority date: 2023-10-24
Filing date: 2024-10-22
Publication date: 2025-05-01
Anticipated expiration: 2026-04-24

Abstract

The invention concerns modified recombinant BLG proteins and uses thereof.

Description

MILK PROTEIN SUBSTITUTE AND PRODUCTS MADE THEREFROM

TECHNOLOGICAL FIELD

The invention generally contemplates milk protein substitutes and products made therefrom.

BACKGROUND

Dairy-like food products are typically derived from vegetarian or vegan sources. While their use is fueled by a variety of commercial and dietary reasons, these products have demonstrated a disappointing degree of commercial acceptance. Some of the factors responsible for the reduced consumer attraction include appearance, flavor and/or mouth feel. Further, many of the available dairy substitutes present lower nutritional values than their dairy equivalents. While production of dairy substitutes from other natural sources may be desirable, presence of certain materials limit their application in food products.

REFERENCES

[1] Japanese Patent Application, Laid-Open (Kokai), No. 2003-250460,

[2] US Patent Application No. 2007/0254065.

GENERAL DESCRIPTION

To improve on the commercial appearance and user experience of dairy products, the inventors of the technology disclosed herein have embarked on the development and manufacture of a modified recombinant beta-lactoglobulin (mrBLG). Products formed of the novel mrBLG of the invention, unlike products formed of recombinant BLG or BLG, demonstrated a dramatic improvement not only in the appearance of the products as nonanimal dairy products and diary substitutes, but also a great improvement in their creaminess, foaming, and stability, endowing them with a longer shelf-life and increased user satisfaction.

In most general terms, the mrBLG is formed by chemically or physically modifying a recombinant beta-lactoglobulin (rBLG) that is substantially free of bovine milk or whey proteins. The modification process converts the recombinant BLG into a material that is distinguishable from the unmodified rBLG and the BLG in its activity, stability, as well as in its analytical signature or characteristics. As demonstrated herein, the mrBLG obtained by the chemical/physical routes disclosed herein exhibited improved protein properties and effectiveness in providing dairy-like products that closely resemble both the visuality and organoleptic properties of real dairy products.

P-Lactoglobulin (BLG) is a major whey protein of cow and sheep's milk. Several variants of BLG are known. The two main variants are identified as isoform A and isoform B. Bovine BLG is a relatively small protein of 162 residues, with a molecular weight reflective of the isoforms: 18.28 or 18.37 kDa for isoform A and isoform B, respectively. BLG occurs in monomeric, dimeric, tetrameric, octameric and other multimeric aggregation forms under a variety of natural conditions. The tendency to aggregate varies at different pHs, salt concentrations, buffer types, and other varied conditions.

Generally, the recombinant protein can be produced through the manipulation of gene expression in an organism by fusing sequences of foreign DNA into a host cell. The altered DNA is then inserted into the host genome, after which it can be replicated, transcribed, and translated to a recombinant protein and further purified. The selection of a suitable expression system and/or host organism depends on the desired production scale and the intended use of the recombinant protein, whereas the choice of vector is largely governed by the host.

Both cell-based and cell-free technologies are available for expressing recombinant proteins. Cell-based systems can be eukaryotic and prokaryotic systems. Recombinant protein production in mammalian cells can be conducted transiently or through stable cell lines. While stable cell lines can be used reproducibly, transient production offers higher-yields. Recombinant protein production in yeast is also an efficient strategy for producing large amounts of the recombinant proteins with high levels of purity. Proteins expressed have a certain degree of the post-translational processing capacity of mammalian proteins and are more stable than prokaryotic systems. Several yeast species can be used for recombinant protein expression, including Saccharomyces, Schizosaccharomyces pombe, Pichia pastoris and Hansanuela polymorpha. Fungal and bacterial expression systems for producing the recombinant protein are also used because of their simple operation, high expression level, and short culture period. For example, the rBLG in a highly purified form (>85% of total protein) may be produced by fermentation of a fungal strain Aspergillus oryzae. In cell-free systems, protein synthesis may be carried out in vitro using translation-compatible extracts of whole cells that contain all the components needed for transcription, translation, and even post-translational modification. With additional supplements of cofactors, proteins of interest can even be formed in a matter of hours.

In addition to the aforementioned methods of production, the recombinant protein may be obtained commercially (available from evitria, ACRObiosymthesis and others).

As used herein, the term “modified” or any lingual variation thereof, refers to a protein that is derived from recombinant BLG (rBLG), which has undergone a structural change that improves its properties, as disclosed herein, rendering it suitable for a variety of uses. The modified recombinant BLG (mrBLG) has been formed from both rBLG isoform A and isoform B, as well as from a mixture of the isoforms. Isoform A having a CAS no. 50863-92-8 is of SEQ ID NO: 1; and isoform B having a CAS no. 9066-45-9 is of SEQ ID NO: 2. Modified rBLG may be similarly obtained from a mixture of the two isoforms (said mixture having a CAS no. 9045-23-2).

LIVTQT MKGLDIQKVA GTWYSLAMAA SDISLLDAQS APLRVYVEEL KPTPEGDLEI LLQKWENDEC AQKKIIAEKT KIPAVFKIDA LNENKVLVLD TDYKKYLLFC MENSAEPEQS LVCQCLVRTP EVDDEALEKF DKALKALPMH IRLSFNPTQL EEQCHI* (SEQ ID NO: 1)

LIVTQT MKGLDIQKVA GTWYSLAMAA SDISLLDAQS APLRVYVEEL KPTPEGDLEI LLQKWENGEC AQKKIIAEKT KIPAVFKIDA LNENKVLVLD TDYKKYLLFC MENSAEPEQS LACQCLVRTP EVDDEALEKF DKALKALPMH IRLSFNPTQL EEQCHI* (SEQ ID NO: 2)

As may be noted, the two sequences differ in positions 64 (D^G) and 118 (V A).

Processes for achieving the mrBLG product of the invention have been developed and have been fine-tuned so that the product may be provided in commercial quantities, in a reproducible manner and in sufficient purity. Without wishing to be bound by theory, processes disclosed herein for providing the mrBLG may involve a plurality of inter- and intra- molecular interactions that convert the rBLG into a random peptide mixture that comprises a mixture of charged and neutral proteins, monomers and multimers of different sizes, chemically interacting proteins wherein protein monomers are associated through, e.g., S-S bonds or H-binding, and aggregated proteins. Typically, the formed mixture comprises molecular species of different molecular weight, being same or similar or higher than a molecular weight of the unmodified rBLG. The formed mixture may be further treated, or may be used as is, without isolation of specific components of the mixture. Despite the fact that the random peptide mixture comprises numerous protein forms and other components, as disclosed herein, the mixture is well characterized and distinguishable from both the recombinant BLG and the bovine BLG.

As used herein, the mrBLG is a "random peptide mixture, RPM" comprising a unique mixture of modified rBLG peptides manufactured as disclosed herein. The degree of modification may be undefined as over the modification period and modification conditions, different regions of the protein backbone may be modified or may be involved in the chemical or physical modification. It is nevertheless believed that the RPM comprises a mixture of modified proteins, having different degrees of modification. Despite the fact that the exact content of the RPM is not of importance, the content may be determined by acceptable methodologies for determining protein structures and for determining ratio concentrations of each protein type. As is further discussed below, the RPM may be characterized by SEC-HPLC, providing a reliable and coherent method of qualitative characterization.

The mrBLG may be a deamidated rBLG (herein d-rBLG) wherein one or a plurality of glutamine residues (Q) of the rBLG is/are deamidated to provide the corresponding glutamic acid residues (E). Thus, the deamidated rBLG derived from isoform A may be of SEQ ID NO: 3, or when derived from isoform B may be of SEQ ID NO: 4, wherein each of Xi through X9, independently for each of the isoforms, may be Q or E, provided that at least one of said Xi through X9 is a deamidated residue- E.

LIVTXiT MKGLDIX2KVA GTWYSLAMAA SDISLLDAX3S APLRVYVEEL KPTPEGDLEI LLX4KWENDEC AX5KKIIAEKT KIPAVFKIDA LNENKVLVLD TDYKKYLLFC MENSAEPEX₆S LVCX7CLVRTP EVDDEALEKF DKALKALPMH IRLSFNPTXsL EEX9CHI* (SEQ ID NO: 3).

LIVTXiT MKGLDIX2KVA GTWYSLAMAA SDISLLDAX3S APLRVYVEEL KPTPEGDLEI LLX4KWENGEC AX5KKIIAEKT KIPAVFKIDA LNENKVLVLD TDYKKYLLFC MENSAEPEX₆S LACX7CLVRTP EVDDEALEKF DKALKALPMH IRLSFNPTXsL EEX9CHI* (SEQ ID NO: 4).

In some embodiments, the d-rBLG of the invention may be of SEQ ID NO: 3 or SEQ ID NO: 4, wherein, independently for each sequence, at least one, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8 or all 9 of the variables Xi through X9 is E.

In some embodiments, the d-rBLG of the invention may be of SEQ ID NO: 3, wherein at least one of the variables Xi through X9 is E.

In some embodiments, the d-rBLG of the invention may be of SEQ ID NO: 3, wherein at least 2 of the variables Xi through X9 is E.

In some embodiments, the d-rBLG of the invention may be of SEQ ID NO: 3, wherein at least 3 of the variables Xi through X9 is E.

In some embodiments, the d-rBLG of the invention may be of SEQ ID NO: 3, wherein at least 4 of the variables Xi through X9 is E.

In some embodiments, the d-rBLG of the invention may be of SEQ ID NO: 3, wherein at least 5 of the variables Xi through X9 is E.

In some embodiments, the d-rBLG of the invention may be of SEQ ID NO: 3, wherein at least 6 of the variables Xi through X9 is E.

In some embodiments, the d-rBLG of the invention may be of SEQ ID NO: 3, wherein at least 7 of the variables Xi through X9 is E.

In some embodiments, the d-rBLG of the invention may be of SEQ ID NO: 3, wherein at least 8 of the variables Xi through X9 is E.

In some embodiments, the d-rBLG of the invention may be of SEQ ID NO: 3, wherein all of variables Xi through X9 is E.

In some embodiments, the d-rBLG of the invention may be of SEQ ID NO: 4, wherein at least one of the variables Xi through X9 is E.

In some embodiments, the d-rBLG of the invention may be of SEQ ID NO: 4, wherein at least 2 of the variables Xi through X9 is E.

In some embodiments, the d-rBLG of the invention may be of SEQ ID NO: 4, wherein at least 3 of the variables Xi through X9 is E.

In some embodiments, the d-rBLG of the invention may be of SEQ ID NO: 4, wherein at least 4 of the variables Xi through X9 is E. In some embodiments, the d-rBLG of the invention may be of SEQ ID NO: 4, wherein at least 5 of the variables Xi through X9 is E.

In some embodiments, the d-rBLG of the invention may be of SEQ ID NO: 4, wherein at least 6 of the variables Xi through X9 is E.

In some embodiments, the d-rBLG of the invention may be of SEQ ID NO: 4, wherein at least 7 of the variables Xi through X9 is E.

In some embodiments, the d-rBLG of the invention may be of SEQ ID NO: 4, wherein at least 8 of the variables Xi through X9 is E.

In some embodiments, the d-rBLG of the invention may be of SEQ ID NO: 4, wherein all of variables Xi through X9 is E.

In some embodiments, the d-rBLG of the invention is derived from both SEQ ID NO: 3 and SEQ ID NO: 4 and may comprise a mixture of proteins wherein at least one, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8 or all 9 of the variables Xi through X9 of either or both sequences is/are E.

In some embodiments, the d-rBLG is a random peptide mixture (RPM) comprising a plurality of deamidated rBLG proteins of SEQ ID NO: 3, and/or SEQ ID NO: 4, wherein in the protein having SEQ ID NO: 3 or SEQ ID NO: 4 at least one, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8 or all 9 of variables Xi through X9 is E.

In some embodiments, the RPM is a mixture of deamidated rBLG peptides synthesized by random deamidation of a rBLG sample. The degree of deamidation is undefined as the over the processing period, different positions along the protein backbone (defined as Xi through X9) may have different rates of deamidation, or may be differently approached by the deamidating agent or differently responsive to the deamidation conditions. It is nevertheless believed that the RPM comprises a mixture of deamidated proteins, having different degrees of deamidation. Some may have a single deamidated position (any one of positions Xi through X9), while others may have at least 2, 3... or more of the positions deamidated. As used herein, the expression "at least 2, at least 3, at least 4..., ...at least 8..." and so forth signifies a mixture of proteins of SEQ ID NO: 3 , or SEQ ID NO: 4, wherein the mixture may comprise:

-a mixture of different proteins having each at least 1 of variables Xi through X9 being E, and all other variables Xi through X9 being Q; and/or -a mixture of different proteins having each at least 2 of variables Xi through X9 being E, and all other variables Xi through X9 being Q; and/or

-a mixture of different proteins having each at least 3 of variables Xi through X9 being E, and all other variables Xi through X9 being Q; and/or

-a mixture of different proteins having each at least 4 of variables Xi through X9 being E, and all other variables Xi through X9 being Q; and/or

-a mixture of different proteins having each at least 5 of variables Xi through X9 being E, and all other variables Xi through X9 being Q; and/or

-a mixture of different proteins having each at least 6 of variables Xi through X9 being E, and all other variables Xi through X9 being Q; and/or

-a mixture of different proteins having each at least 7 of variables Xi through X9 being E, and all other variables Xi through X9 being Q; and/or

-a mixture of different proteins having each at least 8 of variables Xi through X9 being E, and all other variables Xi through X9 being Q; and/or

-a mixture of different proteins having at least 1 and/or at least 2, and/or at least 3, and/or at least 4, and/or at least 5, and/or at least 6, and/or at least 7, and/or at least 8, and/or all of variables Xi through X9 being E, and all remaining variables Xi through X9 being Q.

In some embodiments, the d-rBLG as defined and exemplified herein is not a contamination product nor an opportunistic protein that may be present in contaminating amounts with rBLG. Rather, in some embodiments, the d-rBLG is formed by enzymatic deamidation, e.g., using glutaminase.

The d-rBLG of the invention may be produced under enzymatic conditions. As exemplified, the rBLG may be treated with e.g., a glutaminase, at a temperature and for a period of time sufficient to achieve the transformation of the rBLG to the d-rBLG, as defined herein. In some embodiments, deamidation was achieved under enzymatic conditions at a temperature between 20 and 60°C for a period of 0.5 to 200 minutes.

The modification of the rBLG molecular weight, charge density, form or structure may be additionally or alternatively achieved by microparticulation, acid treatment, high shear or by titration, as further disclosed. Each of these methods yields a product that is characterized similarly or identically to the d-rBLG of the invention and produces product characteristics that are essentially comparable or same. In some embodiments, the mrBLG is achievable by microparticulation to generate a microparticulated r-BLG, herein referred to as “mp-rBLG”. Microparticulation may be achieved by a variety of methods, such as thermochemical means involving extrusion techniques, e.g., with the aid of a twin-screw extrusion technology. Utilizing a twin-screw extruder configured with a long barrel, a rBLG content of about 5 and 20%, a barrel temperature between 60 and 110°C, a screw rotation speed of between 20 and 300 rpm and feed rate of 1-30 kg/h, a modified rBLG in a form of microparticulates having a diameter between 0.1 and 50 pm may be achieved. Additionally or alternatively, spray drying may be used. Spray drying may be carried out, transforming the same into microparticles.

Alternatively, microparticulation may involve fast change in temperature followed by an increase in the pressure of the sample and fast cooling. Other methods of microparticulation may also be used.

It should be clear that microparticulation, constituting a modified rBLG (mrBLG) of the invention renders unmodified rBLG different in its particulate dimensions and exhibits a SEC-HPLC chromatogram as described herein.

In some embodiments, the mrBLG is obtained through high shear treatment of rBLG under low pH conditions. The mrBLG obtained by this method is referred to herein as “hs-rBLG”. The high shear conditions involve use of a ultrafiltration (UF) diafiltration (DF) system. Multiple UF/DF operations are repeated over a period of more than 20 hours (a time period ranging between hours to weeks). In a typical UF/DF cycle the rBLG is circulated at pH 3.0+1 at a transmembrane pressure (TMP) of between 1.25 and 3.5 bars. The membrane may be a hollow fiber, a tubular, a flat sheet or spiral wound membrane of a polymer selected from PES (polyethersulfone), PVDF (polyvinylidenedifluoride), and PAN (polyacrylonitrile), with a molecular weight cut-off from 1,000 Da and 500,000 Da. In some embodiments, the membrane is a PES spiral-wound membrane.

It should be clear that the rBLG treated under high shear conditions at a low pH, constituting a modified rBLG (mrBLG) of the invention differs from the unmodified rBLG in its particulate dimensions and exhibits a SEC-HPLC chromatogram as described herein.

In some embodiments, the mrBLG is obtained by titration of an acidic formulation of rBLG with a base. The mrBLG obtained by titration under basic conditions is referred to herein as “tt-rBLG”. In the titration method, the rBLG is maintained at a pH 3.0+1 to pH 7.2+0.8 and titrated with a base (e.g., at a concentration equal to or about, or greater thanlM NaOH). The rBLG is adjusted from pH 3.0+1 to pH 7.2+0.8 with a base, such as a NaOH solution, of a molarity of about IM, under high flux conditions, with slow mixing, so as to maximize localization of the base in the rBLG solution. The mrBLG obtained through base titration may be characterized by dimerization or intermolecular interactions through generation of S-S and/or H-bonding and other physical interactions. It should be clear that the rBLG treated under basic conditions, constituting a modified rBLG (mrBLG) of the invention differs from the unmodified rBLG in its particulate dimensions and exhibits a SEC-HPLC chromatogram as described herein.

Modified rBLG products of the invention may be in a form selected from d-rBLG, mp-rBLG, hs-rBLG, and tt-rBLG.

Notwithstanding the type of modification used for producing a mrBLG, the mrBLG may be distinguished from bovine BLG and from an unmodified rBLG by a multitude of spectrophotometric and/or chromatographic techniques. One such technique is size-exclusion HPLC chromatography (SEC-HPLC). As demonstrated in Figs. 1A-C, the mrBLG monomer exhibits a unique peak pattern at a retention time indicative of a molecular weight ranging between 18.36 and 2,000 KDa. As compared with both unmodified BLG and unmodified rBLG (herein the unmodified controls) and demonstrated in Figs. 1A-C, the unique peak pattern of the mrBLG between protein markers 18.36 KDa and 2,000 KDa distinguishes the mrBLG from that of the unmodified controls. The marker at 18.36 kDa is marked with a vertical dotted line in each of Figs. 1A-C. The protein markers are standard markers, chosen based on their molecular weights to support high resolution of the SEC-HPLC.

A correlation between the molecular weight and retention volumes in the SEC- HPLS are demonstrated in Fig. ID.

Thus, in a first of its aspects, the invention concerns a modified recombinant BLG (mrBLG) protein characterized by a size-exclusion HPLC chromatogram (SEC-HPLC) including a peak pattern at a retention time between a protein marker of 18.36 kDa and a protein marker of 2,000 kDa, wherein the mrBLG is provided substantially free of bovine milk or whey proteins.

The invention further provides a deamidated recombinant BLG (d-rBLG) monomer having SEQ ID NO: 3 or SEQ ID NO: 4, as defined herein. The invention also provides a random peptide mixture (RPM) of randomly deamidated rBLG proteins, each of the proteins having SEQ ID NO: 3 or SEQ ID NO: 4, as defined herein.

In some embodiments, the d-rBLG monomer is characterized by a size-exclusion HPLC chromatogram (SEC-HPLC) having a peak pattern at a retention time between a protein marker of 18.36 kDa and a protein marker of 2000 kDa.

The invention further provides a modified recombinant BLG (mrBLG) monomer in a form of a microparticulated rBLG characterized by a size-exclusion HPLC chromatogram (SEC-HPLC) having a peak pattern at a retention time between a protein marker of 18.36 kDa and a protein marker of 2000 kDa, wherein the mrBLG is provided substantially free of bovine milk or whey proteins.

In some embodiments, the microparticulated rBLG is formed by a thermochemical step followed by spray drying.

In some embodiments, the microparticulated rBLG is in a form of microparticles having a size or a diameter between 0.1 and 50 pm.

In some embodiments, the modified recombinant BLG (mrBLG) monomer is in a form of microparticles having a size or a diameter between 0.1 and 50 pm and characterized by a size-exclusion HPLC chromatogram (SEC-HPLC) having a peak pattern at a retention time between a protein marker of 18.36 kDa and a protein marker of 2,000 kDa.

Each of the modified rBLG of the invention, namely d-rBLG, mp-rBLG, hs-rBLG and tt-rBLG, is characterized by a peak pattern at a retention time window between a protein marker of 18.36 kDa and a protein marker of 2000 kDa in the SEC-HPLC chromatogram thereof. The peak pattern, being a feature that a person skilled in the art would not attribute to background noise, comprises a plurality of peaks that are not present in the SEC-HPLC chromatograms obtained for the unmodified controls (e.g., rBLG) under the same conditions. The peak pattern comprises two or more visible independent peak structures, suggesting presence of a plurality of protein species or a plurality of different forms of the modified proteins. The material may include residual host cell proteins, unmodified BLG, dimeric forms, tetrameric forms and others.

It should be noted that the any discussion herein regarding the SEC-HPLC chromatogram of the mrBLG excludes from the scope of the invention any of the unmodified materials. The region of the chromatogram characterized by the peak pattern, as disclosed herein, is unique to the mrBLG of the invention and is not characteristic of the unmodified forms. The full chromatogram of the mrBLG comprises additional peaks or an additional peak pattern below 18.36 kDa. However, as this region of the chromatogram is not by itself indicative of the mrBLG materials, since it overlaps with features that can be attributed to the unmodified forms, this region of the chromatogram (below 18.36 kDa) is not used herein to distinguish between the modified and unmodified materials (namely between mrBLG of the invention and BLG or rBLG). Putting it differently, the SEC-HPLC chromatogram of the mrBLG comprises a peak pattern between retention times corresponding to protein markers 18.36 kDa and 2000 kDa and a peak pattern below 18.36 kDa, while the unmodified forms have SEC-HPLC chromatograms free of peak patterns between 18.36 kDa and 2000 kDa.

The term “peak patern used herein has the general meaning in the art. The peak pattern is a feature or a collection of peaks of same or different intensities and shapes that a person skilled in the art would not attribute to background noise, but would attribute to a protein component, e.g., different mrBLG, monomeric or oligomeric forms and so forth. The peak pattern in the SEC-HPLC chromatogram of the rBLG between a protein marker of 18.36 kDa and a protein marker of 2000 kDa, is indicative of the presence of mrBLG, as defined herein. The chromatogram thus indicates that the mrBLG has a molecular weight between 18.36 and 2000 kDa. between 18.36 and 1500 kDa, between 18.36 and 1000 kDa, between 18.36 and 950 kDa, between 18.36 and 900 kDa, between 18.36 and 850 kDa, between 18.36 and 800 kDa, between 18.36 and 750 kDa, between 18.36 and

700 kDa, between 18.36 and 650 kDa, between 18.36 and 600 kDa, between 18.36 and

550 kDa, between 18.36 and 500 kDa, between 18.36 and 450 kDa, between 18.36 and

400 kDa, between 19 and 2000 kDa, between 20 and 2000 kDa, between 22 and 2000 kDa, between 24 and 2000 kDa, between 26 and 2000 kDa, between 28 and 2000 kDa, between 30 and 2000 kDa, between 35 and 2000 kDa, between 40 and 2000 kDa, between 45 and 2000 kDa, between 50 and 2000 kDa, between 55 and 2000 kDa, between 60 and 2000 kDa, between 65 and 2000 kDa, between 70 and 2000 kDa, between 75 and 2000 kDa, between 80 and 2000 kDa, between 85 and 2000 kDa, between 90 and 2000 kDa, between 95 and 2000 kDa, between 100 and 2000 kDa, between 150 and 2000 kDa, between 200 and 2000 kDa, between 250 and 2000 kDa, between 300 and 2000 kDa, between 350 and 2000 kDa, between 400 and 2000 kDa, between 450 and 2000 kDa, between 500 and 2000 kDa, between 550 and 2000 kDa, or between 600 and 2000 kDa. In some embodiments, the mrBLG has a molecular weight between 18.36 and 600 kDa, between 19 and 600 kDa, or between 20 and 2000 kDa.

In some embodiments, the mrBLG has a molecular weight between 19 and 600 kDa.

The chromatograms obtained for the different mrBLG monomers of the invention and the chromatograms obtained for the unmodified controls may be obtained on a liquid chromatography system (HPLC/UPLC) Vanquish UltiMate 3000, ThermoScientific. Any other similar and commercially available unit may be similarly used. The size exclusion column may be any such known column, for example Acquity UPLC Protein BEH SEC column, 125 A, 1.7 pm, 4.6x300 mm (Waters) and Acquity UPLC Protein BEH SEC guard column 125 , 1.7 pm, 4.6x30 mm (Waters).

The operating conditions employed were:

Elution buffer: 5 mM Trisodium citrate pH 6.9 ± 0.1;

Flow: 0.2 ml/min;

Column temperature: 30°C;

Detection UV Wavelength X = 280mn; and

Elution time: 26 min.

Additional conditions employed included:

Weak Needle Wash: 90/10 water/methanol;

Strong Needle Wash: 90/10 water/methanol;

Seal Wash: 90/10 water/methanol;

Injection Type: Full loop; and

Injection loading: 6 pg BLG on column.

In some embodiments, the modified recombinant BLG (mrBLG) of the invention (e.g., d-rBLG, mp-rBLG, hs-rBLG and tt-rBLG) is characterized by a size-exclusion HPLC chromatogram (SEC-HPLC) peak pattern at a retention time between a protein marker of 18.36 kDa and a protein marker of 2000 kDa, wherein the SEC-HPLC chromatogram is obtained with a liquid chromatography system equipped with a size exclusion column maintained at 30°C and a UV detector operating at 280nm; wherein elution conditions include an elution buffer being 5 mM trisodium citrate pH 6.9 ± 0.1; flow rate being 0.2 ml/min; and elution time being 26 minutes.

The mrBLG of the invention may be provided in a solid form, in a semi- solid or gel form, in a solution form, or in any commercially suitable form or in combination with any food-grade carrier. In some embodiments, the mrBLG is provided in a solid form or in a suspension form. In other embodiments, the mrBLG is provided in a solution form, e.g., in water or an aqueous medium.

In some embodiments, the mrBLG is provided as a powder. A stable powder of the mrBLG may be obtained by lyophilization or by spray drying or by any other drying technology, as known in the art.

In some embodiments, the d-rBLG is provided in a powder form.

The invention further provides use of a mrBLG for the preparation of a food or a diary product, as disclosed herein.

Further provided is a composition comprising a mrBLG of the invention and at least one solid or a liquid carrier. In some embodiments, the solid or liquid carrier is a food-grade carrier. The food carrier may be selected amongst such materials that are fit for human consumption. Such carriers are considered GRAS (Generally Recognized as Safe). Non-limiting examples of food-grade carriers include water, aqueous media, oils, alcohols, a solid carrier such as a gum or sugar and others.

In some embodiments, the composition comprises a random peptide mixture of at least one mrBLG protein selected from d-rBLG, mp-rBLG, hs-rBLG and tt-rBLG, each as defined herein. In some embodiments, the composition comprises d-rBLG. In some embodiments, the composition comprises mp-rBLG. In some embodiments, the composition comprises hs-rBLG. In some embodiments, the composition comprises tt- rBLG.

Typically, the mrBLG monomer of the invention is obtained free of bovine milk or whey proteins. Such milk or whey proteins may be, but not limited to, butyrophilin subfamily 1, member Al; perilipin 2; kininogen-1; alpha-2-HS-glycoprotein; fatty-acid- binding protein 3; fatty-acid-binding protein 5; beta-2-microglobulin; glycosylationdependent cell adhesion molecule 1, lactophorin; complement factor C3; apolipoprotein Al; apolipoprotein A2; apolipoprotein A4; and apolipoprotein E.

In some embodiments, the mrBLG is provided substantially free of any one or more of butyrophilin subfamily 1, member Al; perilipin 2; kininogen-1; alpha-2-HS- glycoprotein; fatty-acid-binding protein 3; fatty-acid-binding protein 5; beta-2- microglobulin; glycosylation-dependent cell adhesion molecule 1, lactophorin; complement factor C3; apolipoprotein Al; apolipoprotein A2; apolipoprotein A4; and apolipoprotein E. The term “substantially free " means that the milk and/or whey proteins are not detectable in presence of the mrBLG monomer when examined by acceptable analytical methods, or are present in such trace amounts that are not functional; namely, not to materially contribute to properties of the food product comprising the trace amount of the proteins. In some cases, the mrBLG is free, namely having a zero amount of one or more milk and/or whey proteins.

In some embodiments, compositions of the invention are free or substantially free of milk and/or whey proteins, as defined.

Compositions of the invention comprise the mrBLG, a carrier and optionally one or more inert or functional additives. The additive may be a food component such as oils, fats, carbohydrates (e.g., sugars, such as monosaccharides as glucose or fructose; or disaccharides such as sucrose; or oligosaccharides such as maltodextrin), proteins, free amino acids, vitamins (such as vitamin Bl, vitamin B2, vitamin B3, vitamin B5, vitamin B6, vitamin B12, vitamin C, and folate), minerals (such as calcium, magnesium, phosphate, sodium, potassium, and zinc), mineral acids, soluble and insoluble fibers (such as inulin, fructo oligosaccharides, galacto-oligosaccharides, polydextrose, resistant dextrin/maltodextrin and/or beta-glucan rich fibres), flavorings (such as vanilla and chocolate), coloring agents, emulsifiers (e.g., having HLB range from 0 to 25 such as mono-di glycerides, sucrose ester, lecithin), stabilizers, antioxidants, starch (e.g., such as potato, tapioca, corn, wheat), gums (such as carrageenan, guar gum and/or xanthan gum), lecithin, pectin, and others.

In some embodiments, the composition comprises a sugar selected from dextrose, trehalose, sucrose, fructose, lactose, maltodextrin, and others.

In some embodiments, the composition is in a form of a powder. In some embodiments, the composition is in a form of a solution.

In some embodiments, a solution of the mrBLG, e.g., d-rBLG, is treated with at least one sugar. The sugar may be added to the solution immediately after modification of the rBLG, e.g., deamidation, or at any stage prior to solidification or drying of the solution.

The sugar-to-mrBLG w/w ratio may vary based on the final product to be produced. In some embodiments, the suganmrBLG ratio is in favor of the mrBLG. In some embodiments, the ratio is in favor of the sugar. In some embodiments, the suganmrBLG weight ratio is between 0.2:1 and 1.5:1. In some embodiments, the composition comprises d-rBLG and at least one sugar; or comprises mp-rBLG and at least one sugar; or comprises hs-rBLG and at least one sugar; or comprises tt-rBLG and at least one sugar.

The composition comprising the mrBLG may be the reaction mixture in which the mrBLG is formed. This reaction mixture may comprise unmodified rBLG and one or more materials, reactants or side products of the modification reaction.

In some embodiments, the composition comprises mrBLG and unmodified rBLG. The composition may comprise one or more of the additives disclosed herein, e.g., a sugar. Where the composition comprises both mrBLG and unmodified rBLG, the amount of the mrBLG is between about 1 and 50 wt%.

The mrBLG when provided in any form, i.e., solution, dispersion or as a powder, may be used as a raw material or as an additive in the food industry. The mrBLG may be formulated as a composition suitable for human or animal consumption. The composition may be a food composition or a food additive or may generally be regarded as a commercial food component. Such compositions may comprise one or more carrier or solubilizing materials, e.g., solids or liquids, as well as one or more additives as acceptable in the field.

A “food composition comprising mrBLG encompasses all forms of functional foods, nutritional supplements, health foods, animal foods and others that are provided in various forms according to a common knowledge in the art. Non-limiting examples of food products that may comprise mrBLG include vegetable protein-rich feeds; and dairy products.

In some embodiments, the food composition comprising the mrBLG is a dairy product.

The mrBLG of the invention may be provided as a “food additive , namely a material component/compo sition that is provided in a form that is suitable for incorporation in a foodstuff to confer a physical (e.g., consistency, creaminess, color, etc), nutritional, and/or a health benefit to a subject consuming same. Unlike food compositions which may be manufactured with mrBLG as one of its components, when used as a food additive, the mrBLG may be added as is or formulated into a fully formed food composition. The amount of the mrBLG may vary based on the product to incorporate the material. For example, the amount of the material intended to modify the technical characteristics of the food product may be different than the amount used for increasing the nutritional benefit of the food product.

Irrespective of the way by which the mrBLG is incorporated into a food product, food products of the invention may comprise mrBLG and may be substantially free of bovine proteins. The food product may be any such product suitable for human or animal consumption. In some embodiments, the product is a substitute diary product (dairy-like product). In some other embodiments, the product is a non-dairy food product.

The invention provides a dairy-like product (or a non-animal milk product) comprising mrBLG, wherein the dairy-like product is free of bovine proteins. In some embodiments, the diary-like product comprises d-rBLG and/or mp-rBLG and/or hs-rBLG and/or tt-rBLG, each as defined herein.

The dairy-like products of the invention may comprise, in addition to mrBLG, one or more food components such as oils, fats, carbohydrates (e.g., sugars, such as monosaccharides as glucose or fructose; or disaccharides such as sucrose; or oligosaccharides such as maltodextrin), proteins, free amino acids, vitamins (such as vitamin Bl, vitamin B2, vitamin B3, vitamin B5, vitamin B6, vitamin B 12, vitamin C, and folate), minerals (such as calcium, magnesium, phosphate, sodium, potassium, and zinc), mineral acids, soluble and insoluble fibers (such as inulin, fructo oligosaccharides, galacto-oligosaccharides, polydextrose, resistant dextrin/maltodextrin and/or beta-glucan rich fibres), flavorings (such as vanilla and chocolate), coloring agents, emulsifiers (e.g., having HLB range from 0 to 25 such as mono-di glycerides, sucrose ester, lecithin), starch (e.g., such as potato, tapioca, corn, wheat), gums (such as carrageenan, guar gum and/or xanthan gum), lecithin, pectin, and others.

In some embodiments, the additive is a sugar as defined and selected herein.

The "dairy-like product" of the invention may be a milk substitute, milk beverages, yogurt, low-fat yogurt, nonfat yogurt, Greek yogurt, high protein yogurt, whipped yogurt, Labneh, cream cheese, yogurt drinks, sauces, spreads, cream (such as light cream, cooking cream, whipping cream, coffee whitener/creamer, sour cream), frozen confections, desserts, milk protein concentrate and others.

In some embodiments, the dairy-like product is milk; yogurt such as set, stirred, high-protein, sour cream and drinkable yogurt; ice cream and soft-serve ice cream; cheese such as cream, soft, semi-hard and hard cheeses; high-protein dairy beverages; dairy desserts such as various puddings, and custards; whipping and cooking cream. Any of the aforementioned dairy-like products constitutes a separate embodiment of the invention.

The dairy-like product may comprise various amounts of the mrBLG. The amount may depend, inter alia, on the product, the method of its preparation, the effect desired and any other factor known to a person versed in the art. Generally speaking, the amount of the mrBLG may be between about 0.01 and 50 wt% (of the total weight of the product). In some embodiment, the amount of mrBLG is selected so that the total amount of proteins in the product may be between about 0.01 and 50 wt%.

The invention further provides a dairy-like product comprising d-rBLG having a SEQ ID NO: 3 and/or a d-rBLG having a SEQ ID NO: 4, wherein the food product optionally comprises one or more additional proteins, one or more oils and a carbohydrate.

In some embodiments, the product is a yogurt.

In some embodiments, the mrBLG is d-rBLG.

The invention further provides a method of manufacture a deamidated recombinant BLG (d-rBLG), the method comprising treating a recombinant BLG under enzymatic conditions suitable for deamidating one or a plurality of glutamine functionalities of the recombinant BLG. In some embodiments, the rBLG is an isoform A or isoform B or a mixture of rBLG isoforms. In some embodiments, the d-rBLG is of SEQ ID NO: 3 or SEQ ID NO: 4. In some embodiments, the d-rBLG is a mixture of modified isoforms A and B.

Further provided is a kit or a commercial package comprising a recombinant BLG or a modified form thereof and instructions of use.

In some embodiments, the kit or commercial package comprises a recombinant BLG and instructions for forming a modified form thereof, as defined herein. In some embodiments, the kit may further comprise a glutaminase enzyme.

In some embodiments, the kit or commercial package comprises mrBLG, as defined, and instructions for forming a dairy-like product.

The invention provides:

A modified recombinant BLG (mrBLG) protein characterized by a size-exclusion HPLC chromatogram (SEC-HPLC) including a peak pattern at a retention time between a protein marker of 18.36 kDa and a protein marker of 2,000 kDa, wherein the mrBLG is provided substantially free of bovine milk or whey proteins. In some configurations of a mrBLG of the invention, it has a molecular weight between 18.36 and 2,000 kDa or a molecular weight between 19 and 600 kDa.

In some configurations of a mrBLG of the invention, it is a mixture of proteins.

In some configurations of a mrBLG of the invention, the SEC-HPLC chromatogram thereof having a peak pattern below the protein marker of 18. 36 kDa.

In some configurations of a mrBLG of the invention, it is a modified isoform A of rBLG, isoform B of rBLG or a mixture of the isoforms, wherein isoform A is of SEQ ID NO: 1; and isoform B having being of SEQ ID NO: 2.

In some configurations of a mrBLG of the invention, it is a random peptide mixture, RPM, comprising a mixture of mrBLG proteins having different degrees of modification and/or varying molecular weights.

In some configurations of a mrBLG of the invention, it is derived from rBLG by mechanical or chemical treatment.

In some configurations of a mrBLG of the invention, the mechanical or chemical treatment comprises one or more of deamidation, acidification, base treatment, ultra filtration, application of shear forces, and microparticulation.

In some configurations of a mrBLG of the invention, it is a RPM comprising a mixture of deamidated rBLG (d-rBLG) wherein one or a plurality of glutamine residues (Q) of the rBLG is/are deamidated to glutamic acid residues (E).

In some configurations of a mrBLG of the invention, it is a RPM comprising a mixture of proteins being each of SEQ ID NO: 3, and/or a mixture of proteins being each of SEQ ID NO: 4.

In some configurations of a mrBLG of the invention, the mixture of proteins of SEQ ID NO: 3 comprises proteins having amino acid E in at least one of variables Xi through X9 and amino acid Q in the remaining variables.

In some configurations of a mrBLG of the invention, the mixture of proteins of SEQ ID NO: 3 comprises proteins having amino acid E in at least 2 of variables Xi through X9 and amino acid Q in the remaining variables.

In some configurations of a mrBLG of the invention, the mixture of proteins of SEQ ID NO: 3 comprises proteins having amino acid E in least 3 of variables Xi through X9 and amino acid Q in the remaining variables. In some configurations of a mrBLG of the invention, the mixture of proteins of SEQ ID NO: 3 comprises proteins having amino acid E in at least 4 of variables Xi through X9 and amino acid Q in the remaining variables.

In some configurations of a mrBLG of the invention, the mixture of proteins of SEQ ID NO: 3 comprises proteins having amino acid E in at least 5 of variables Xi through X9 and amino acid Q in the remaining variables.

In some configurations of a mrBLG of the invention, the mixture of proteins of SEQ ID NO: 3 comprises proteins having amino acid E in at least 6 of variables Xi through X9 and amino acid Q in the remaining variables.

In some configurations of a mrBLG of the invention, the mixture of proteins of SEQ ID NO: 3 comprises proteins having amino acid E in at least 7 of variables Xi through X9 and amino acid Q in the remaining variables.

In some configurations of a mrBLG of the invention, the mixture of proteins of SEQ ID NO: 3 comprises proteins having amino acid E in at least 8 of variables Xi through X9 and amino acid Q in the remaining variable.

In some configurations of a mrBLG of the invention, the mixture of proteins of SEQ ID NO: 3 comprises proteins having amino acid E in all of variables Xi through X9.

In some configurations of a mrBLG of the invention, the mixture of proteins of SEQ ID NO: 4 comprises proteins having amino acid E in at least one of variables Xi through X9 and amino acid Q in the remaining variables.

In some configurations of a mrBLG of the invention, the mixture of proteins of SEQ ID NO: 4 comprises proteins having amino acid E in in at least 2 of variables Xi through X9 and amino acid Q in the remaining variables.

In some configurations of a mrBLG of the invention, the mixture of proteins of SEQ ID NO: 4 comprises proteins having amino acid E in at least 3 of variables Xi through X9 and amino acid Q in the remaining variables.

In some configurations of a mrBLG of the invention, the mixture of proteins of SEQ ID NO: 4 comprises proteins having amino acid E in at least 4 of variables Xi through X9 and amino acid Q in the remaining variables.

In some configurations of a mrBLG of the invention, the mixture of proteins of SEQ ID NO: 4 comprises proteins having amino acid E in at least 5 of variables Xi through X9 and amino acid Q in the remaining variables. In some configurations of a mrBLG of the invention, the mixture of proteins of SEQ ID NO: 4 comprises proteins having amino acid E in at least 6 of variables Xi through X9 and amino acid Q in the remaining variables.

In some configurations of a mrBLG of the invention, the mixture of proteins of SEQ ID NO: 4 comprises proteins having amino acid E in at least 7 of variables Xi through X9 and amino acid Q in the remaining variables.

In some configurations of a mrBLG of the invention, the mixture of proteins of SEQ ID NO: 4 comprises proteins having amino acid E in at least 8 of variables Xi through X9 and amino acid Q in the remaining variable.

In some configurations of a mrBLG of the invention, the mixture of proteins of SEQ ID NO: 4 comprises proteins having amino acid E in all of variables Xi through X9.

In some configurations of a mrBLG of the invention, the mixture of proteins of SEQ ID NO: 3 and/or SEQ ID NO: 4 being a mixture of proteins wherein at least one, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8 or all 9 of variables Xi through X9 of either or both sequences is E, and amino acid Q in the remaining variables.

In some configurations of a mrBLG of the invention, it is a microparticulated mrBLG.

In some configurations of a mrBLG of the invention, the microparticulated mrBLG being in a form of microparticulates having a diameter between 0.1 and 50 pm.

In some configurations of a mrBLG of the invention, it is obtained by microparticulation.

In some configurations of a mrBLG of the invention, it is obtained by a method comprising application of shear forces and use of an ultrafiltration (UF) diafiltration (DF) system.

In some configurations of a mrBLG of the invention, it is obtained by titration of an acidic formulation of rBLG, maintained at a pH 3.0+1 to pH 7.2+0.8 with a base solution.

The invention further provides a modified recombinant BLG (mrBLG) protein in a form of a deamidated or a microparticulated rBLG characterized by a size-exclusion HPLC chromatogram (SEC-HPLC) having a peak pattern at a retention time between a protein marker of 18.36 kDa and a protein marker of 2000 kDa, wherein the mrBLG is provided substantially free of bovine milk or whey proteins. In some configurations of a mrBLG of the invention, it is of SEQ ID NO: 3 or SEQ ID NO:4 or a mixture of mrBLG proteins, at least one of which having a SEQ ID NOG and at least one other having a SEQ ID NO: 4.

In some configurations of a mrBLG of the invention, it is characterized by a sizeexclusion HPLC chromatogram (SEC-HPLC) peak pattern at a retention time between a protein marker of 18.36 kDa and a protein marker of 2000 kDa, wherein the SEC-HPLC chromatogram is obtained with a liquid chromatography system equipped with a size exclusion column.

In some configurations of a mrBLG of the invention, the column is maintained at 30°C and a UV detector operating at 280nm.

In some configurations of a mrBLG of the invention, the system employs elution conditions including an elution buffer being 5 mM trisodium citrate pH 6.9 ± 0.1; flow rate being 0.2 ml/min; and elution time being 26 minutes.

In some configurations of a mrBLG of the invention, it is in a powder form.

In some configurations of a mrBLG of the invention, it is for use in a method of preparing a food or a diary product.

A composition is provided that comprises any mrBLG according to the invention and at least one solid or a liquid carrier.

In some configurations of a composition of the invention, it is a suspension of the mrBLG in an aqueous medium.

In some configurations of a composition of the invention, it comprises a random peptide mixture of the mrBLG.

In some configurations of a composition of the invention, the mrBLG is d-rBLG.

In some configurations of a composition of the invention, it is substantially free of bovine milk or whey proteins.

In some configurations of a composition of the invention, it comprises one or more inert or functional additives.

In some configurations of a composition of the invention, the additives being selected amongst oils, fats, carbohydrates, proteins, free amino acids, vitamins, minerals, mineral acids, soluble and insoluble fibers, flavorings, coloring agents, emulsifiers, stabilizers, antioxidants, starch, lecithin, and pectin.

In some configurations of a composition of the invention, it comprises at least one sugar. In some configurations of a composition of the invention, the at least one sugar is selected from dextrose, trehalose, sucrose, fructose, lactose, and maltodextrin.

In some configurations of a composition of the invention, the sugar-to-mrBLG w/w ratio is between 0.2:1 and 1.5:1.

In some configurations of a composition of the invention, it is a food composition.

In some configurations of a composition of the invention, it is a vegetable proteinrich feed or a dairy product.

In some configurations of a composition of the invention, it comprises rBLG.

In some configurations of a composition of the invention, the mrBLG constitutes at least lwt% of a protein content.

A dairy-like product (or a non-animal milk product) is provided that comprises mrBLG according to the invention or a composition according to the invention, wherein the dairy-like product is free of bovine proteins.

In some configurations of a product of the invention, it comprises rBLG.

In some configurations of a product of the invention, it is a milk substitute, milk beverage, yogurt, low-fat yogurt, nonfat yogurt, Greek yogurt, high protein yogurt, whipped yogurt, Labneh, cream cheese, yogurt drinks, sauces, spreads, cream (such as light cream, cooking cream, whipping cream, coffee whitener/creamer, sour cream), frozen confections, desserts, or milk protein concentrate .

In some configurations of a product of the invention, it comprises mrBLG in an amount being at least 0.5 wt% (of the total weight of the product).

Further provided is a dairy-like product comprising d-rBLG having a SEQ ID NO: 3 or SEQ ID NO: 4, or a mixture thereof, wherein the product optionally comprises one or more additional proteins, one or more oils and a carbohydrate.

A mixture is provided which comprises at least one mrBLG according to the invention and rBLG, wherein the rBLG is of SEQ ID NO: 1 or 2.

In some configurations of a mixture of the invention, the mrBLG is of SEQ ID NO: 3 or 4.

In some configurations of a mixture of the invention, the rBLG is of SEQ ID NO:

1 and said mrBLG is of SEQ ID NO: 3.

In some configurations of a mixture of the invention, the rBLG is of SEQ ID NO:

2 and said mrBLG is of SEQ ID NO: 4. In some configurations of a mixture of the invention, it is for use in formulating a food product.

A kit or a commercial package is also provided which comprises a mixture according to the invention, and instructions of use.

BRIEF DESCRIPTION OF THE DRAWINGS

In order to better understand the subject matter that is disclosed herein and to exemplify how it may be carried out in practice, embodiments will now be described, by way of non-limiting example only, with reference to the accompanying drawings, in which:

Figs. 1A-D: SEC-HPLC chromatograms showing a modified recombinant BLG and an unmodified rBLG/BLG. Fig. 1A: SEC-HPLC chromatogram of d-rBLG as compared with unmodified rBLG. Fig. IB: SEC-HPLC of different batches of tt-rBLG as compared to unmodified rBLG and bovine BLG. Fig. 1C: SEC-HPLC of hs-rBLG as compared to unmodified bovine BLG. A vertical dotted line designates the marker molecular weight of 18.36 kDa. Fig. ID: Correlation between molecular weight and normalized retention volumes in SEC-HPLC chromatograms of mrBLG products of the invention. The horizontal bar indicates a molecular weight of 18.36 kDa. The 2,000 kDa mark is not shown.

Figs. 2A-B: Milk beverages 24 hours after preparation. Fig. 2A: a milk beverage based on unmodified rBLG, and Fig. 2B: a sample containing an amount of d-rBLG .

Figs. 3A-B: Coffee coagulation test. Fig. 3A: a coffee formed containing unmodified rBLG, and Fig. 3B: a sample containing d-rBLG.

Fig. 4: Foaming capacity (%) and foaming stability (measured 5 min after whipping) of milk beverages (from left: 0= unmodified rBLG, 40=containing d-rBLG ).

Figs. 5A-C: provide results of Fig. 5A: sensory evaluations of yogurt prepared with an unmodified rBLG (reference) and modified d-rBLG. The score range was between 1 and 5, with 1 being the least creamy; Fig. 5B: appearance of the yogurt when inserting a spoon into a sample containing unmodified rBLG (reference), and Fig. 5C: with d-rBLG.

Fig. 6: Improvement of yogurt texture through use of sugars as spray-dry excipients. Yogurts prepared from: (A) unmodified rBLG, (B) and (C) deamidated rBLG at 2 different concentrations (4U/g and lOU/g, respectively) and spray-dried without use

RECTIFIED SHEET (RULE 91) of sugars, (D) deamidated rBLG with lOU/g PG and spray-dried with 1:1 ratio of dextrose, (E) deamidated rBLG with lOU/g PG and spray-dried with 1:1 ratio of sucrose.

DETAILED DESCRIPTION OF EMBODIMENTS

Deamidation of recombinant beta-lactoglobulin improves creaminess of set and stirred yogurts (4% protein) as well as high-protein yogurt (up to 10% protein).

When deamidated recombinant beta-lactoglobulin is used to prepare a non-animal milk beverage for barista use, deamidation prevents coagulation in coffee, without the need for additional emulsifiers, stabilizers, and acidity regulators. In addition, deamidation of recombinant beta-lactoglobulin improves foaming properties of the milk beverage for barista use: a higher volume of foam is produced, and the foam stability over time is improved.

Materials And Methods

Materials

A recombinant BLG served as BLG source. Protein-glutaminase “Amano”500 was obtained from Amano Enzyme Inc., Japan. Dextrose Monohydrate was purchased from Baolingbao Biology Co., Ltd (BLB), China, and Qiqihar Longjiang Fufeng Biotechnologies Co. Ltd., China. Trehalose was supplied by Shandong Fuyang Bio-tech Co. Ltd., China. Sucrose was obtained from Sugat, Israel. Lactose 200 monohydrate was manufactured by DMV-Fonterra Excipients GmbH & Co. KG, Germany. Gellan Gum LT- 100 was purchased from CP-Kelco Ltd, Denmark. Sunflower oil was purchased from AAK Ltd, Sweden. Salt was purchased from Sugat, Israel. Bacterial Culture Vege 053 and Vege 047 were purchased from Danisco, Denmark. Tri-sodium citrate dihydrate and P-lactoglobulin from bovine milk were obtained from Sigma.

Manufacture of rBLG

Host strain: A. oryzae strain RIB40, a filamentous fungus, is used as a base strain in the construction of the production strain of P-lactoglobulin (strain Ao_st0002). A. oryzae is a Biosafety Level 1 organism, as classified by the American Type Culture Collection, and has a long-history of safe use in the production of food enzymes. The production strain A. oryzae Ao_st0002 was constructed from the recipient host strain A. oryzae Ao_st0044. The recipient host strain A. oryzae Ao_st0044 was constructed from the base strain A. oryzae RIB40 by deletion of the endogenous orotidine-5'- monophosphate decarboxylase gene (pyrG) and LigD. Deletion of pyrG results in an uracil auxotrophic Strain Ao_st0044 as the gene pyrG is an established homolog to URA3 in Saccharomyces cerevisiae. Therefore, uracil was used as a selection marker for Strain Ao_st0044. The A. oryzae strain RIB40 has been deposited in an international culture collection, World Data Centre for Microorganisms (WDCM), as WDCM 139 and was obtained from a previous collection of microorganisms in 2019.

Construction of the Production Strain: Genetic modification practices that are commonly used and well defined were employed in order to obtain both a pure product and optimize expression of P-lactoglobulin. A. oryzae is frequently used as a production organism because of its characterized secretion capacity, genetic tractability, and fast growth. The Ao_st0002 production strain was constructed by transformation of 2 or more expression cassettes to the recipient strain Ao_st0044. The expression cassette consisted of a nucleic acid sequence of the P-lactoglobulin gene from the domestic cow (Bos taurus) fused to a secretion signal flanked by a promoter and a terminator sequence. The fusion of the secretion signal was designed with the addition of a cleavage site, removing the secretion signal with no trace of the signal or the cleavage site. Thus, the resulting amino acid sequence was identical to the bovine P-lactoglobulin isoform B protein. The nucleic acid sequence of P-lactoglobulin was optimized to induce maximal levels of expression without changing the native amino acid sequence of the protein. The expression cassette also contains the coding sequence of the Aspergillus niger orotidine-5 '-monophosphate decarboxylase gene (pyrA) homolog of the A. oryzae pyrG (GenBank: X96734.2). pyrA is utilized to regain the uracil production used as a selection marker from the pyrG deleted auxotrophic Ao_st0044 strain. In addition, the expression cassette contains a series of well characterized transcriptional elements such as promoters, terminators, and transcription factors to promote the expression of the P-lactoglobulin gene from endogenous elements of non-toxigenic relative species or synthetic sources. No antibiotic selection markers or origin of replication sequences were used in the construction process of the production strain Ao_st0002 or the recipient host strain Ao_st0044.

Genomic Stability of the Production Strain: A multi-generational genomic stability study was conducted of the production strain. Genomic stability was assessed by growing the fungi from spores of the production strain on solid media plates until the production of new spores. Spores of Ao_st0002 were collected (representing a new generation of the fungi) and placed on a fresh solid media plate to grow. Genomic DNA was isolated from the spores and copy number of the P-lactoglobulin gene was evaluated by quantitative polymerase chain reaction (qPCR). The copy-number of the P- lactoglobulin gene in the fifth generation was evaluated and compared to the copynumber of the mother strain (generation 0) by the qPCR delta-delta (2-AACT ) method. The results show a consistent copy-number of the P-lactoglobulin gene after 5 generations of the fungi with RQ = 1.109 ± 0.031 (normalized relative quantity), establishing a genetic stability of Ao_st0002 production strain for 5 generations.

Manufacturing Process: the rBLG was manufactured by precision fermentation of genetically modified species of A. oryzae without expression induction by solvents such as methanol. The fermentation process began with the insertion of spore suspension stock vial seed into the seed fermentation stage to increase biomass amount. At the end of the seed fermentation stage, the culture was transferred to the main fermentation stage to produce P-lactoglobulin. Following the fermentation stage, the biomass was separated from the broth. The harvest then underwent a series of purification steps: pH, conductivity, and temperature adjustment (optional); centrifugation and/or filtration to remove impurities; concentration and dialysis using ultrafiltration/diafiltration; sterile filtration; and spray drying. The final product was a white to off-white to yellowish powder consisting of >60% total protein, of which rBLG was not less than 85%.

Deamidation of recombinant BLG to obtain a modified rBLG: d-rBLG

A recombinant BLG was resuspended in water to a final protein concentration of 30-400 mg/ml. PG500 enzyme (a protein glutaminase) was added at concentrations of 0.5 -100 U/g. The solution was then mixed and incubated in a pre-heated water bath or jacketed reactor maintained at a temperature between 40 and 55 °C for a period of 5 to 140 minutes prior to processing into a dairy product or prior to spray-drying as detailed below.

Microparticulation of rBLG to obtain a modified rBLG: mp-BLG

Using a SPX LeanCreme device, 5-20% rBLG is “predenaturated” in a controlled manner at a temperature between 60 and 85°C and is circulated at high pressure for few seconds to few minutes. Subsequently, the sample is cooled by fast cooling to 4°C. The product is then spray dried at inlet temperature of 150-200°C and outlet temperature of 50-90°C.

High shear treatment of rBLG at low pH for a period of days to several weeks to obtain a modified rBLG: hs-rBLG

A rBLG was circulated at pH 3.0+1 for a period of 20h or more, in a UF/DF system comprising a PES spiral-wound membrane and a transmembrane pressure (TMP) of 1.25 and 3.5 bars, generating a shear force on the protein treated.

Titration from pH 3.0+1 to pH 7.2+0.8 with a high local base concentration to obtain a modified r-BLG: tt-rBLG

A rBLG water solution was adjusted from a pH 3.0+1 to pH 7.2+0.8 with the addition of a IM NaOH solution, at high flux under slow mixing so as not to cause distribution of the base in the solution. This resulted in a high local NaOH concentration in a region of the BLG solution where the NaOH solution was added.

Dissolution of sugars with a modified rBLG

Various sugars such as dextrose, trehalose, sucrose, and lactose were dissolved in the mrBLG solution, immediately after the modification process was completed, e.g., immediately after the deamidation was finished and while the deamidation solution was still warm, or at any other time prior to drying, e.g., spray-drying, at w/w ratios between 0.2:1 and 1.5:1 of sugar:BLG. Thereafter, the mixture of the modified BLG and the sugar(s) was dried, lyophilized or spray-dried.

Characterization of modified rBLG through SEC-HPLC

Deamidated as well as other modified rBLG samples were analyzed on the liquid chromatography system (HPLC/UPLC) Vanquish UltiMate 3000, ThermoScientific with an Acquity UPLC Protein BEH SEC column, 125 A, 1.7 pm, 4.6x300 mm (Waters) and an Acquity UPLC Protein BEH SEC guard column 125 , 1.7 pm, 4.6x30 mm (Waters). The method parameters employed were:

Elution buffer: 5 mM Trisodium citrate pH 6.9 + 0.1

Flow: 0.2 ml/min

Column T°: 30°C Detection UV Wavelength X = 280mn

Elution time: 26 min

Weak Needle Wash: 90/10 water/methanol

Strong Needle Wash: 90/10 water/methanol

Seal Wash: 90/10 water/methanol

Injection Type: Full loop

Injection loading: 6 pg BLG on column

Under similar or identical conditions, all mrBLG products of the invention may be analyzed.

Food Applications Preparation

Yogurt Preparation

An exemplary yogurt formulation is provided in Table 1:

The yogurt was prepared by adding the mixed dry ingredients into water at room temperature. The mixture was hydrated for 40 minutes. The solution was then preheated to 65°C and vegetable oil added and homogenized using a Homogenizer (APV Lab 2000, SPX Flow) at 200+50 bar. Then the solution was pasteurized at 85°C for 5 minutes using a digital ceramic plate (Selmor, SE435) and rapidly cooled with cold water to 42°C. Bacterial culture was then added and the solution was fermented until pH reached 4.2- 4.5. Yogurt was then stirred until texture was smooth, and set in the fridge, 4°C, for at least 24h before tasting.

Milk Beverage Preparation

An exemplary beverage is provided in Table 2:

All dry ingredients were mixed and added to preheated water 40°C and mixed at speed 4 for 30 seconds in a Thermomix (Vorwerk, TM5). Then the solution mixed for additional 10 minutes at speed 2. Sunflower oil was added and speed increased to 4 for additional 40 seconds. Then solution was pasteurized: 85°C for 2 minutes. Afterwards, the solution cooled to 60°C and was homogenized using 2 stages: 200+50 bar (Homogenizer APV Lab 2000, SPX Flow).

Milk Beverage Tests

Foaming Capacity and Stability (FC & FS).

A measured volume of each milk beverage sample was foamed using a Milk Frother (Universe, NRI-947). The volume of the foam received was recorded using a graduated cylinder and the foaming capacity was calculated as follows:

Volume increase (%) = (V2-Vl/Vl)*100

Vl= initial volume of milk solution; V2= volume of milk solution after foaming.

For the determination of FS, foam volume changes in the graduated cylinder were recorded after 5 minutes. Coagulation in Coffee

The milk beverage was added to coffee that was prepared using Platinum Brazil coffee (Elite) and boiling water. The test was made by pouring 20% cold milk beverage (4°C) to 80% boiling coffee (85°C). The coagulation test was measured visually.

Sensorial test for Yogurt

A group of 5 trained tasters evaluated the Creaminess texture of the yogurt.

Milk Beverages, with applications in coffee creaming

Improved emulsification: Milk beverages prepared with deamidated rBLG without addition of emulsifiers and stabilizers do not show the phase separation seen in the reference sample (see Error! Reference source not found.) 24 hours after preparation. This proves that modification improves the emulsification properties of rBLG. Enzymatic treatment, therefore, is a method that allows the preparation of “clean label” milk beverages that do not contain emulsifiers and stabilizers or use reduced amount of the same. Improved emulsification increases the storage stability of milk beverages and is advantageous also in many other applicational aspects.

Inhibited precipitation and curdling at low pH, without use of acidity regulators: deamidated rBLG does not precipitate and curdle in coffee, as opposed to the un-treated reference. Error! Reference source not found, shows that modified sample inhibited coagulation (measured pH was 4.8) compared to the untreated BLG. All samples were prepared without emulsifiers, stabilizers, and acidity regulators. The lack of a need for acidity regulators allows preparation of “clean label” coffee creamers and barista milk beverages through rBLG modification.

Improved Foaming for barista applications

The foaming capacity expresses the increase in foam volume immediately after whipping ((V2-Vi)/Vi*100), V2 (ml): volume after whipping, Vi (ml): initial volume). Modification clearly increases foaming capacity at all enzyme concentrations.

Samples were prepared without emulsifiers and stabilizers. Yogurt

Great increase in creaminess: deamidated modified rBLG forms yogurt with a very smooth and creamy texture, while the reference yogurt is stiff gel and gelatine-like texture. This can be seen in Error! Reference source not found.. Creaminess was evaluated with in-house sensory panels: Error! Reference source not found. A a show the results of sensory evaluation. The scoring range was 1-5, with 5 being the creamiest. We conclude from the sensory evaluations that modification greatly improves creaminess of yogurt.

Sugars as spray-drying excipients for modified BLG preserve creaminess and smoothness of the yogurts

The dissolution of various sugars in the deamidated solutions prior to spray-drying preserves the smooth and creamy texture of the yogurts prepared from the resulting powders. Yogurts made from spray-dried powders of modified BLG with sugars as excipients show smoothness and creaminess similar to yogurts made from freshly prepared modified solutions (see Fig. 1, D. and E.). On the other hand, yogurts prepared from modified BLG that had been spray-dried without addition of sugars are less smooth and tend to form small clumps (see Fig. 1, B. and C.).

SEC-HPLC chromatograms of modified BLG show 2 characteristic features that correlate with creamy texture of yogurts: a slight left- shift of the main peak towards higher molecular weight and an increase in the peak area of the higher molecular weight components (dimer, tetramer,. .

Claims

CLAIMS:

1. A modified recombinant BLG (mrBLG) protein characterized by a size-exclusion HPLC chromatogram (SEC-HPLC) including a peak pattern at a retention time between a protein marker of 18.36 kDa and a protein marker of 2,000 kDa, wherein the mrBLG is provided substantially free of bovine milk or whey proteins.

2. The mrBLG of claim 1, having a molecular weight between 18.36 and 2,000 kDa or a molecular weight between 19 and 600 kDa.

3. The mrBLG according to claim 1 or 2, being a mixture of proteins.

4. The mrBLG according to any one of the preceding claims, wherein the SEC- HPLC chromatogram thereof having a peak pattern below the protein marker of 18. 36 kDa.

5. The mrBLG according to any one of claims 1 to 4, being a modified isoform A of rBLG, isoform B of rBLG or a mixture of the isoforms, wherein isoform A is of SEQ ID NO: 1; and isoform B having being of SEQ ID NO: 2.

6. The mrBLG according to any one of claims 1 to 5, being a random peptide mixture, RPM, comprising a mixture of mrBLG proteins having different degrees of modification and/or varying molecular weights.

7. The mrBLG according to any one of the preceding claims, derived from rBLG by mechanical or chemical treatment.

8. The mrBLG according to claim 7, wherein the mechanical or chemical treatment comprises one or more of deamidation, acidification, base treatment, ultra filtration, application of shear forces, and microparticulation.

9. The mrBLG according to any one of the preceding claims, the mrBLG being a RPM comprising a mixture of deamidated rBLG (d-rBLG) wherein one or a plurality of glutamine residues (Q) of the rBLG is/are deamidated to glutamic acid residues (E).

10. The mrBLG according to any one of the preceding claims, the mrBLG being a RPM comprising a mixture of proteins being each of SEQ ID NO: 3, and/or a mixture of proteins being each of SEQ ID NO: 4.

11. The mrBLG according to claim 10, the mixture of proteins of SEQ ID NO: 3 comprises proteins having amino acid E in at least one of variables Xi through X9 and amino acid Q in the remaining variables.

12. The mrBLG according to claim 10, the mixture of proteins of SEQ ID NO: 3 comprises proteins having amino acid E in at least 2 of variables Xi through X9 and amino acid Q in the remaining variables.

13. The mrBLG according to claim 10, the mixture of proteins of SEQ ID NO: 3 comprises proteins having amino acid E in least 3 of variables Xi through X9 and amino acid Q in the remaining variables.

14. The mrBLG according to claim 10, the mixture of proteins of SEQ ID NO: 3 comprises proteins having amino acid E in at least 4 of variables Xi through X9 and amino acid Q in the remaining variables.

15. The mrBLG according to claim 10, the mixture of proteins of SEQ ID NO: 3 comprises proteins having amino acid E in at least 5 of variables Xi through X9 and amino acid Q in the remaining variables.

16. The mrBLG according to claim 10, the mixture of proteins of SEQ ID NO: 3 comprises proteins having amino acid E in at least 6 of variables Xi through X9 and amino acid Q in the remaining variables.

17. The mrBLG according to claim 10, the mixture of proteins of SEQ ID NO: 3 comprises proteins having amino acid E in at least 7 of variables Xi through X9 and amino acid Q in the remaining variables.

18. The mrBLG according to claim 10, the mixture of proteins of SEQ ID NO: 3 comprises proteins having amino acid E in at least 8 of variables Xi through X9 and amino acid Q in the remaining variable.

19. The mrBLG according to claim 10, the mixture of proteins of SEQ ID NO: 3 comprises proteins having amino acid E in all of variables Xi through X9.

20. The mrBLG according to claim 10, the mixture of proteins of SEQ ID NO: 4 comprises proteins having amino acid E in at least one of variables Xi through X9 and amino acid Q in the remaining variables.

21. The mrBLG according to claim 10, the mixture of proteins of SEQ ID NO: 4 comprises proteins having amino acid E in in at least 2 of variables Xi through X9 and amino acid Q in the remaining variables.

22. The mrBLG according to claim 10, the mixture of proteins of SEQ ID NO: 4 comprises proteins having amino acid E in at least 3 of variables Xi through X9 and amino acid Q in the remaining variables.

23. The mrBLG according to claim 10, the mixture of proteins of SEQ ID NO: 4 comprises proteins having amino acid E in at least 4 of variables Xi through X9 and amino acid Q in the remaining variables.

24. The mrBLG according to claim 10, the mixture of proteins of SEQ ID NO: 4 comprises proteins having amino acid E in at least 5 of variables Xi through X9 and amino acid Q in the remaining variables.

25. The mrBLG according to claim 10, the mixture of proteins of SEQ ID NO: 4 comprises proteins having amino acid E in at least 6 of variables Xi through X9 and amino acid Q in the remaining variables.

26. The mrBLG according to claim 10, the mixture of proteins of SEQ ID NO: 4 comprises proteins having amino acid E in at least 7 of variables Xi through X9 and amino acid Q in the remaining variables.

27. The mrBLG according to claim 10, the mixture of proteins of SEQ ID NO: 4 comprises proteins having amino acid E in at least 8 of variables Xi through X9 and amino acid Q in the remaining variable.

28. The mrBLG according to claim 10, the mixture of proteins of SEQ ID NO: 4 comprises proteins having amino acid E in all of variables Xi through X9.

29. The mrBLG according to claim 10, the mixture of proteins of SEQ ID NO: 3 and/or SEQ ID NO: 4 being a mixture of proteins wherein at least one, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8 or all 9 of variables Xi through X9 of either or both sequences is E, and amino acid Q in the remaining variables.

30. The mrBLG according to any one of claims 1 to 8, being a microparticulated mrBLG.

31. The mrBLG according to claim 30, wherein the microparticulated mrBLG being in a form of microparticulates having a diameter between 0.1 and 50 pm.

32. The mrBLG according to any one of claims 1 to 8, obtained by microparticulation.

33. The mrBLG according to any one of claims 1 to 8, obtained by a method comprising application of shear forces and use of an ultrafiltration (UF) diafiltration (DF) system.

34. The mrBLG according to any one of claims 1 to 8, obtained by titration of an acidic formulation of rBLG, maintained at a pH 3.0+1 to pH 7.2+0.8 with a base solution.

35. A modified recombinant BLG (mrBLG) protein in a form of a deamidated or a microparticulated rBLG characterized by a size-exclusion HPLC chromatogram (SEC- HPLC) having a peak pattern at a retention time between a protein marker of 18.36 kDa and a protein marker of 2000 kDa, wherein the mrBLG is provided substantially free of bovine milk or whey proteins.

36. The mrBLG according to claim 35, being of SEQ ID NO: 3 or SEQ ID NO: 4 or a mixture of mrBLG proteins, at least one of which having a SEQ ID NOG and at least one other having a SEQ ID NO: 4.

37. The mrBLG according to any one of the preceding claims, characterized by a sizeexclusion HPLC chromatogram (SEC-HPLC) peak pattern at a retention time between a protein marker of 18.36 kDa and a protein marker of 2000 kDa, wherein the SEC-HPLC chromatogram is obtained with a liquid chromatography system equipped with a size exclusion column.

38. The mrBLG according to claim 37, wherein the column is maintained at 30°C and a UV detector operating at 280nm.

39. The mrBLG according to claim 37 or 38, wherein the system employs elution conditions including an elution buffer being 5 mM trisodium citrate pH 6.9 ± 0.1; flow rate being 0.2 ml/min; and elution time being 26 minutes.

40. The mrBLG according to any one of the preceding claims, in a powder form.

41. Use of a mrBLG according to any one of claims 1 to 40, in a method of preparing a food or a diary product.

42. A composition comprising a mrBLG according to any one of claims 1 to 41 and at least one solid or a liquid carrier.

43. The composition according to claim 42, being a suspension of the mrBLG in an aqueous medium.

44. The composition according to claim 42 or 43, comprising a random peptide mixture of the mrBLG.

45. The composition according to claim 42 to 44, wherein the mrBLG is d-rBLG.

46. The composition according to any one of claims 42 to 45, being substantially free of bovine milk or whey proteins.

47. The composition according to any one of claims 42 to 46, comprising one or more inert or functional additives.

48. The composition according to claim 47, wherein the additives being selected amongst oils, fats, carbohydrates, proteins, free amino acids, vitamins, minerals, mineral acids, soluble and insoluble fibers, flavorings, coloring agents, emulsifiers, stabilizers, antioxidants, starch, lecithin, and pectin.

49. The composition according to any one of claims 42 to 48, comprising at least one sugar.

50. The composition according to claim 49, wherein the at least one sugar is selected from dextrose, trehalose, sucrose, fructose, lactose, and maltodextrin.

51. The composition according to claim 49 or 50, wherein the sugar-to-mrBLG w/w ratio is between 0.2:1 and 1.5:1.

52. The composition according to any one of claims 42 to 51, being a food composition.

53. The composition according to claim 52, being a vegetable protein-rich feed or a dairy product.

54. The composition according to any one of claims 42 to 53, comprising rBLG.

55. The composition according to any one of claims 42 to 54, wherein the mrBLG constitutes at least lwt% of a protein content.

56. A dairy-like product (or a non-animal milk product) comprising mrBLG according to any one of claims 1 to 40 or a composition according to any one of claims 42 to 55, wherein the dairy-like product is free of bovine proteins.

57. The product according to claim 56, comprising rBLG.

58. The product according to claim 56 or 57, being a milk substitute, milk beverage, yogurt, low-fat yogurt, nonfat yogurt, Greek yogurt, high protein yogurt, whipped yogurt, Labneh, cream cheese, yogurt drinks, sauces, spreads, cream (such as light cream, cooking cream, whipping cream, coffee whitener/creamer, sour cream), frozen confections, desserts, or milk protein concentrate .

59. The product according to any one of claims 56 to 58, comprises mrBLG in an amount being at least 0.5 wt% (of the total weight of the product).

60. A dairy-like product comprising d-rBLG having a SEQ ID NO: 3 or SEQ ID NO: 4, or a mixture thereof, wherein the product optionally comprises one or more additional proteins, one or more oils and a carbohydrate.

61. A mixture comprising at least one mrBLG according to any one of claims 1 to 40 and rBLG, wherein the rBLG is of SEQ ID NO: 1 or 2.

62. The mixture according to claim 61, wherein the mrBLG is of SEQ ID NO: 3 or 4.

63. The mixture according to claim 61, wherein the rBLG is of SEQ ID NO: 1 and said mrBLG is of SEQ ID NO: 3.

64. The mixture according to claim 61, wherein the rBLG is of SEQ ID NO: 2 and said mrBLG is of SEQ ID NO: 4.

65. The mixture according to any one of claims 61 to 64, for use in formulating a food product.

66. A kit or a commercial package comprising a mixture according to any one of claims 61 to 64, and instructions of use.