Use of serine proteases for improving the digestibility of high protein dairy products

Abstract

The present invention relates to the preparation of acidified milk products with high protein content, which preparation comprises a treatment with a serine protease.

Description

Technical Field

[0001] This invention relates to the use of proteases in the preparation of acidified dairy products with high protein content. Background Technology

[0002] The demand for protein-rich products, particularly acidified dairy products such as yogurt, is growing, as these products not only provide high-quality protein but also enhanced nutritional functionality. However, protein enrichment can alter the physicochemical and sensory properties of conventional acidified dairy products. Acidification of dairy products can be achieved through the use of lactic acid bacteria fermentation and / or by the direct addition of proteolytic enzymes. Using selected lactic acid bacteria strains (optionally in combination with protease treatment) can enhance the proteolysis of milk proteins, thereby increasing the levels of free amino acids and peptides of different molecular sizes in the final product.

[0003] However, excessive protein hydrolysis can negatively impact the sensory properties of a product, particularly by imparting an unpleasant bitterness and adversely affecting its texture. Therefore, there remains a need for improved acidified dairy products that maintain ideal taste and texture while offering high protein content and enhanced levels of free amino acids and peptides. Summary of the Invention

[0004] The purpose of this invention is to provide acidified dairy products with high protein content that exhibit increased levels of free amino acids and peptides, improved digestibility and nutrient utilization, and desirable sensory profiles, while avoiding bitterness.

[0005] The inventors screened different types of proteases and found that treating the milk base with serine proteases before or during acidification resulted in acidified dairy products with high protein content exhibiting increased levels of free amino acids and peptides, as well as improved digestibility and nutrient utilization. Furthermore, compared to acidified dairy products prepared using the same method but without serine protease treatment, the resulting acidified dairy products exhibited improved texture-related sensory properties, preferably mouth thickness, viscosity, and / or smoothness, without increasing bitterness.

[0006] In one aspect, the present invention relates to Galaya ® Smooth is used to improve the digestibility of protein in fermented dairy products.

[0007] On the other hand, the present invention relates to Galaya ®Smooth is used to increase the concentration of a desired peptide in drinkable yogurt, wherein the desired peptide is NAVPITPTL, preferably wherein, during in vitro digestion according to the INFOGEST protocol, the concentration of the desired peptide is increased by 144%–191% in 8% protein yogurt and by 13%–255% in 12% protein yogurt, relative to an enzyme-free control.

[0008] On the other hand, the present invention relates to Galaya ® Smooth is used to improve texture-related sensory properties, preferably a thick, viscous and / or smooth feel in the mouth, without increasing bitterness.

[0009] On the other hand, the present invention relates to a method for preparing a fermented dairy product containing a protein with improved digestibility, the method comprising using Galaya ® Smooth-treated milk base, wherein the protein content of the milk base is in the range of 4.5 wt%–12 wt%, preferably 8 wt%–12 wt%. Attached Figure Description

[0010] Figure 1 The effect of serine protease addition on the total free amino acid concentration in microyogurt produced using different starter cultures with a total protein content of 10% or 4.5% was described.

[0011] Figure 2 The effects of serine protease addition on the concentration of free essential amino acids in miniature yogurts produced using different starter cultures ((a) Yoflex Creamy 2.0, (b) YF-L904, or (c) Yoflex Premium 1.0) with a total protein content of 10% or 4.5% were described.

[0012] Figure 3 The effects of serine protease addition on the concentrations of free isoleucine, leucine, and lysine in miniature yogurts produced using different starter cultures ((a) Yoflex Creamy 2.0, (b) YF-L904, or (c) Yoflex Premium 1.0) with a total protein content of 10% or 4.5% were described.

[0013] Figure 4 The effect of adding serine protease on the degree of hydrolysis in high-protein yogurts with a total protein content of 8% or 12% was described.

[0014] Figure 5The effects of serine protease addition on peptide formation and abundance in high-protein yogurts with 8% or 12% total protein were described: a) the number of different peptides formed in yogurt with 8% total protein after serine protease addition; b) the intensity (corresponding to abundance) of different peptides formed in yogurt with 8% total protein after serine protease addition; c) the number of different peptides formed in yogurt with 12% total protein after serine protease addition; d) the intensity (corresponding to abundance) of different peptides formed in yogurt with 12% total protein after serine protease addition.

[0015] Figure 6 The effects of serine protease addition on peptide formation and abundance in high-protein yogurts containing 8% or 12% total protein during in vitro digestion with INFOGEST are illustrated. The data presented were obtained at the start of the intestinal phase (corresponding to the in vivo absorption window as defined by the INFOGEST protocol). a) Number of different peptides formed in yogurt with 8% total protein after serine protease addition; b) Intensity (corresponding to abundance) of different peptides formed in yogurt with 8% total protein after serine protease addition; c) Number of different peptides formed in yogurt with 12% total protein after serine protease addition; d) Intensity (corresponding to abundance) of different peptides formed in yogurt with 12% total protein after serine protease addition.

[0016] Figure 7 The effects of adding serine proteases on the formation of the desired peptide NAVPITTTL in high-protein yogurts containing 8% or 12% total protein during in vitro digestion with INFOKEST are demonstrated. Data were obtained at the start of the intestinal phase (corresponding to the beginning of the in vivo absorption phase).

[0017] Figure 8 The effect of adding serine proteases on the formation of protein fragments and larger peptides in high-protein yogurt containing 8% or 12% total protein was demonstrated.

[0018] Figure 9 The effect of the addition of serine protease on the formation of peptides with fewer than 7 amino acids in high-protein yogurt containing 8% total protein during in vitro digestion with INFOGEST was demonstrated, assessed at 0, 15, 30, 60 and 120 min during intestinal phase simulated digestion (representing the time window for in vivo absorption).

[0019] Figure 10 The effect of the addition of serine protease on the formation of peptides with fewer than 7 amino acids in high-protein yogurt containing 12% total protein during in vitro digestion with INFOGEST was demonstrated, assessed at 0, 15, 30, 60 and 120 min during intestinal phase simulated digestion (representing the time window for in vivo absorption).

[0020] Figure 11 The effect of microbial serine protease dosage on shear stress and visual flow properties of fermented dairy products was demonstrated.

[0021] Figure 12 Sensory profile analysis of fermented dairy products containing different doses of microbial serine proteases is shown. Detailed Implementation

[0022] In one aspect, the present invention relates to Galaya ® Smooth is used to improve the digestibility of protein in fermented dairy products.

[0023] In the context of this invention, acidified dairy products refer to milk-based products produced through acidification.

[0024] In one embodiment, acidification is carried out by incubation with lactic acid bacteria, preferably belonging to the genera *Streptococcus*, *Lactococcus*, *Lactobacillus*, *Leuconostoc*, *Pseudobacillus*, *Pediococcus*, *Propionibacterium*, *Enterococcus*, *Bryophyte*, or *Bifidobacterium*, or any combination thereof. Acidification by incubation with one or more lactic acid bacteria can be referred to as fermentation.

[0025] In another embodiment, acidification is carried out by incubation with a chemical acidifying agent (preferably glucono delta-lactone (GDL)).

[0026] In a preferred embodiment, the acidified dairy product is high-protein yogurt, Greek yogurt, Labneh yogurt, skyr yogurt, or sour cream.

[0027] In a preferred embodiment, the protein content of the drinkable yogurt is in the range of 4.5 wt%-12 wt%, preferably 8 wt%-12 wt%.

[0028] Acidified dairy products can be spoonable, such as stirred yogurt or set-type yogurt, or drinkable, such as drinkable yogurt or beverage-type yogurt.

[0029] Stirred yogurt can be produced by acidification in a fermentation tank, where the acidic gel formed after acidification (when the desired pH is achieved) is broken up, for example, by agitation. The stirred product can be partially cooled to 20°C-30°C and flavoring ingredients added. The stirred product is then pumped to a filling line and filled into retail containers. Afterward, the stirred yogurt product can be cooled and then stored.

[0030] Set yogurt can be acidified in retail containers without stirring. After acidification, the set yogurt can be cooled and then stored. Cooling can be carried out in a rapid cooling tunnel or a refrigerator.

[0031] The term "fermented dairy products" includes yogurt. The term "yogurt" typically covers dairy products produced by fermentation with a starter culture containing a combination of Lactobacillus species (such as Lactobacillus bulgaricus) and Streptococcus thermophilus, or any other suitable combination of microorganisms.

[0032] In a preferred embodiment, the fermented dairy product is drinkable yogurt.

[0033] The "drinkable acidified dairy products" according to the invention include any drinkable product based on an acidified milk base, and therefore include fermented milk beverages and liquid yogurt beverages. The drinkable yogurts according to the invention are drinkable, in this sense, they are in liquid form and can be consumed as a beverage, i.e., they are suitable for drinking rather than being scooped with a spoon ("spoonable"). "Liquid form" means that the product is in a fluid state and therefore exhibits a tendency to flow easily. Thus, the shape of a liquid is generally determined by the container it is filled with, unlike, for example, gel-like substances (such as pudding), which are soft but not free-flowing. The drinkable yogurts according to the invention can have a viscosity, thereby allowing consumers to drink the product using a straw as needed.

[0034] As used in this article, the term "post-acidification" refers to the point when acidification is complete and the desired pH is achieved.

[0035] If a chemical acidifier is used, the desired pH is, for example, around pH 4.5.

[0036] If microbial fermentation is used for acidification, the pH after acidification can preferably be between 3.5 and 5.5, and most preferably between 4 and 5.

[0037] In a preferred embodiment, the present invention relates to a method for preparing a fermented dairy product containing a protein with improved digestibility, the method comprising using Galaya... ® Smooth-treated milk base, wherein the protein content of the milk base is in the range of 4.5 wt%–12 wt%, preferably 8 wt%–12 wt%.

[0038] In a preferred embodiment, the product is drinkable yogurt.

[0039] In another preferred embodiment, the present invention relates to a method for preparing dairy products, the method comprising: a) providing a milk substrate; b) treating the milk substrate with an enzyme having serine protease activity; and c) fermenting the milk substrate with microorganisms.

[0040] In the method of this invention, heat treatment (e.g., pasteurization step) can be performed after step (a) and before step (b). Heat treatment can typically be performed at 90°C-95°C for 5 minutes, or at 85°C for 30 minutes, although other possibilities exist.

[0041] In step (b), the treatment with serine protease can be carried out, for example, at 40°C-55°C, such as at 41°C-55°C, for 15 minutes to 10 hours, or for example, for 30 minutes to 3 hours.

[0042] Perform acidification in step (b) until the desired pH is reached. The optimal temperature and incubation time for acidification are well known in the art. Acidification can be performed, for example, at 40°C–45°C for 3–10 hours, or for example, for 4–7 hours. Sometimes, lower temperatures (e.g., as low as 32°C) can be used for mesophilic incubation.

[0043] In a preferred embodiment, steps (b) and (c) can be performed sequentially or simultaneously. The serine endopeptidase and the acidifying agent, i.e., the chemical acidifier and / or lactic acid bacteria, can be added substantially simultaneously. Acidification is performed until the desired pH is reached, for example, at 40°C-45°C for 3-10 hours, or for example, 4-7 hours.

[0044] As used herein, the terms "milk base" or "milk foundation" may cover any milk or dairy product derived from mammals, preferably cow's milk, sheep's milk, or goat's milk, which has been concentrated or fortified to obtain a protein content of 6%-20%, for example 6%-15% (w / w). In one embodiment, the milk base may be derived from whole milk. In a preferred embodiment, the milk base may be derived from low-fat milk, such as 1% fat milk, 0.1% fat milk, semi-skimmed milk, or skimmed milk. The milk base may be reconstituted skim milk powder, casein, whey protein (WPI or WPC), milk protein concentrate, or any combination thereof. The milk base may be any type of milk fortified with skim milk powder, casein, whey protein (WPI or WPC), milk protein concentrate, or any combination thereof. The milk base may also be a concentrated or fortified blended milk. The milk base may be concentrated or fortified UHT milk.

[0045] The milk substrate may be fortified with protein, preferably milk protein, or concentrated to increase its protein content to 6%-20%, for example 6%-15% or 7%-10% (w / w).

[0046] In one embodiment, the emulsion substrate has been concentrated using ultrafiltration prior to acidification.

[0047] Prior to fermentation, the milk substrate can be homogenized and pasteurized using methods known in the art.

[0048] As used in any embodiment of this invention within the context of the invention, "homogenization" means vigorous mixing to obtain a soluble suspension or emulsion. If homogenization is performed prior to fermentation, it can be used to break down milk fat into smaller sizes so that it no longer separates from the milk. This can be accomplished by forcing the milk through small pores under high pressure.

[0049] As used in the context of this invention and in any of its embodiments, "pasteurization" means treating the milk base to reduce or eliminate the presence of living organisms, such as microorganisms. Preferably, pasteurization is achieved by maintaining a specified temperature for a specified time. The specified temperature is typically achieved by heating. The temperature and duration can be selected to kill or inactivate certain bacteria, such as harmful bacteria. A rapid cooling step may then be performed.

[0050] In the context of this invention, in any of its embodiments, "fermentation" means the conversion of carbohydrates into acids or alcohols, or a mixture of both, by the action of microorganisms (LAB). Fermentation processes used in the production of food products (such as dairy products) are well known, and those skilled in the art will know how to select appropriate process conditions, such as temperature, oxygen, the amount of microorganisms, and processing time.

[0051] In a preferred embodiment, the starter culture is (a) Yoflex Creamy 2.0, (b) YF-L904, or (c) Yoflex Premium 1.0, which are commercially available from Chr. Hansen (A / S).

[0052] In a preferred embodiment, the serine protease is Galaya. ® Smooth.

[0053] In a preferred embodiment, Galaya ® Smooth also increases the concentration of free amino acids in drinkable yogurt.

[0054] In another preferred embodiment, Galaya ®Smooth treatment increases the availability of free essential amino acids, including branched-chain amino acids such as leucine, isoleucine, and valine. Leucine is widely considered a key anabolic trigger for muscle protein synthesis, while the remaining essential amino acids serve as substrates to support net protein accumulation and maintain the anabolic response [1-3]. Free amino acids, as well as small peptides, produced by a combination of fermentation and protease treatment, may be utilized more rapidly than intact proteins because they require less or no further luminal proteolysis before intestinal uptake, thus contributing to a faster postprandial appearance of amino acids in circulation [4-7]. The rapid postprandial availability of essential amino acids is associated with a stronger stimulus to muscle protein synthesis, and studies comparing rapidly digested proteins with more slowly digested proteins have shown differences in hyperacidity and postprandial protein handling [4-7].

[0055] By using the serine protease Galaya ® Smooth proteins enable controlled proteolysis, thereby increasing the degree of hydrolysis (DH%) and generating smaller peptides that are more readily digested by human gastrointestinal proteases. Therefore, such pre-hydrolyzed proteins can provide a milder protein matrix with improved digestibility and faster absorption kinetics [4-7]. Furthermore, dipeptides and tripeptides can be directly absorbed by intestinal peptide transporters (e.g., PepT1), which can further promote rapid amino acid uptake compared to intact proteins [8-10].

[0056] In a preferred embodiment, Galaya is used. ® Smooth, with a 50%-120% increase in free amino acid concentration, preferably 70%-120%, corresponds to a level of 29 to 86 mg / 100 mL yogurt in 10% yogurt, compared to a level of 21 to 26 mg / 100 mL yogurt in 4.5% yogurt.

[0057] In another preferred embodiment, by using Galaya ® Smooth, the increase in free amino acid concentration includes an increase in total essential amino acid concentration, corresponding to a free essential amino acid level of 79 to 207 mg / 100 mL yogurt in 10% yogurt and a free essential amino acid level of 48 to 69 mg / 100 mL yogurt in 4.5% yogurt.

[0058] In another preferred embodiment, by using Galaya ® Smooth, the increase of essential amino acids includes an increase in the concentration of lysine, preferably an increase of 10-20 mg / 100 mL in the concentration of lysine.

[0059] In another preferred embodiment, by using Galaya ® Smooth, the increase of essential amino acids includes an increase in the concentration of free leucine, preferably an increase of 2-10 mg / 100 mL in the concentration of free leucine.

[0060] In another preferred embodiment, by using Galaya ® Smooth, the increase of essential amino acids includes an increase in the concentration of free isoleucine, preferably an increase of 2 mg / 100 mL in the concentration of free leucine.

[0061] In another preferred embodiment, Galaya is used. ® Smooth also increases the degree of hydrolysis of drinkable yogurt by 4%-26%.

[0062] In another preferred embodiment, during in vitro digestion according to the INFOGEST protocol, Galaya was used relative to the enzyme-free control. ® Smooth also increases the concentration of peptides with fewer than 7 amino acids in drinkable yogurt, by 6%–57% in 8% protein yogurt and by 6%–62% in 12% protein yogurt.

[0063] On the other hand, the present invention provides Galaya ® Smooth is used to increase the concentration of a desired peptide in drinkable yogurt, wherein the desired peptide is NAVPITPTL, preferably wherein, during in vitro digestion according to the INFOGEST protocol, the concentration of the desired peptide increases by 144%–191% in 8% protein yogurt and by 13%–255% in 12% protein yogurt relative to an enzyme-free control.

[0064] On the other hand, the present invention provides Galaya ® Smooth is used to increase the number and relative abundance of peptides in the range of 6-25 amino acids in drinkable yogurt, preferably increasing the number and relative abundance of peptides in the range of 8, 12, and 21-22 amino acids, relative to an enzyme-free control; or preferably increasing the number and strength of peptides in the range of 6-15 amino acids.

[0065] On the other hand, the present invention relates to Galaya ® Smooth is used to improve texture-related sensory properties, preferably a thick, viscous and / or smooth feel in the mouth, without increasing bitterness.

[0066] Texture (or oral sensation) is the physical and chemical interaction of a product in the mouth and is an aspect of food rheology. Evaluation begins with the initial perception on the palate, continues through the swallowing process, and extends to the aftertaste. The term describes all tactile observations (or sensations occurring in the oral cavity that are related to oral tissues and their sensory states, such as a feeling of being enveloped) associated with texture and texture sensation in the mouth, including the characteristic “fatty sensation” often referred to as a creamy texture (Barnes et al., 1991, Journal of Dairy Science 74:2089-2099; Lawless and Heyman (1999) Sensory evaluation of food: principles and practices. Aspen Publishers, Inc., Gaithersburg, MD).

[0067] "Thickness in the mouth" can be described as the thickness of yogurt when swallowed at a normal to high rate of consumption; a high thickness in the mouth corresponds to a thick product, which takes longer to swallow (Meilgaard, M., Civille, VG, Carr, BT, ed. 1999. Sensory Evaluation Techniques (3rd ed.). New York: CRC Press.).

[0068] serine protease In a preferred embodiment, the serine protease is a microbial serine protease. Using microbial enzymes instead of animal or plant enzymes has advantages because microbial enzymes exhibit broad-spectrum characteristics (optimal pH, temperature, etc.) and can be stably obtained in large quantities and with high purity. Furthermore, enzymes isolated from, for example, animal sources are susceptible to contamination, such as viral particles and / or viral DNA, which is undesirable when the enzyme is used, for example, in food products.

[0069] In a preferred embodiment, the serine protease is Galaya. ® Smooth is commercially available from Novozymes A / S in Denmark.

[0070] Serine proteases may exist outside the cell. Their N-terminus may have a signaling sequence that is cleaved during secretion.

[0071] Serine proteases can be derived from any of the sources mentioned herein. In the context of this invention, the term "derived from" means that the enzyme can be isolated from the organism in which it naturally occurs, i.e., the amino acid sequence of the serine protease is identical to that of the natural polypeptide. The term "derived from" also means that the enzyme can be recombinantly produced in a host organism, and the recombinant enzyme has an amino acid sequence identical to that of the natural enzyme, or has a modified amino acid sequence (e.g., with one or more amino acids that are deleted, inserted, and / or substituted), i.e., the recombinant enzyme is a mutant of the natural amino acid sequence. The meaning of "natural enzyme" includes natural variants. Furthermore, the term "derived from" includes enzymes synthesized, for example, through peptide synthesis. The term "derived from" also covers enzymes that have been modified in vivo or in vitro, for example, through glycosylation, phosphorylation, etc. For recombinant enzymes, the term "derived from" refers to the identity of the enzyme, not the identity of the host organism from which the recombinant enzyme is produced.

[0072] Serine proteases can be obtained from microorganisms using any suitable technique. For example, enzyme preparations can be obtained by fermenting suitable microorganisms and subsequently isolating the endopeptidase preparation from the resulting fermentation broth or microorganism using methods known in the art. Serine proteases can also be obtained using recombinant DNA technology. Such methods typically involve culturing host cells transformed with a recombinant DNA vector containing a DNA sequence encoding a serine protease, and operatively linking the DNA sequence to a suitable expression signal such that the enzyme can be expressed in a culture medium under conditions allowing enzyme expression, and recovering the enzyme from the culture. DNA sequences can also be incorporated into the genome of host cells. The DNA sequence can be genomic, cDNA, or synthetically derived, or any combination thereof, and can be isolated or synthesized according to methods known in the art.

[0073] Serine proteases can be purified. As used herein, the term "purified" encompasses serine protease proteins that are substantially free of insoluble components from the producing organism. The term "purified" also encompasses serine protease proteins that are substantially free of insoluble components from the native organism from which the enzyme was obtained. Preferably, the enzyme can also be separated from some soluble components of the source organism and the culture medium. More preferably, separation can be performed by one or more unit operations: filtration, precipitation, or chromatography.

[0074] Therefore, serine proteases can be purified, i.e., containing only small amounts of other proteins. The expression "other proteins" specifically refers to other enzymes. The term "purified," as used herein, also refers to the removal of other components present in the serine protease-derived cells, particularly other proteins and most particularly other enzymes. A serine protease can be "substantially pure," i.e., free from other components from the organism that produces the serine protease (i.e., the host organism used for recombinant production of the serine protease). Preferably, the serine protease is an enzyme protein preparation that is at least 40% (w / w) pure, more preferably at least 50%, 60%, 70%, 80%, or even at least 90% pure.

[0075] The term "serine protease" includes any auxiliary compound that may be necessary for the enzyme's catalytic activity, such as a suitable receptor or cofactor, which may or may not be naturally present in the reaction system.

[0076] Serine proteases can be in any form suitable for the use discussed, such as as dry powder or granules, dust-free granules, liquids, stabilized liquids, or protected enzymes.

[0077] The serine protease used in the method of the present invention can be added at a concentration of 1-3000 KPRU / kg substrate protein, preferably 5-2000 KPRU / kg substrate protein, and more preferably 25-600 KPRU / kg substrate protein. The dosage will depend on parameters such as temperature and incubation time. Those skilled in the art will know how to determine the optimal enzyme dosage.

[0078] In the method of the present invention, the emulsion substrate is optimally treated only with microbial serine proteases, wherein other proteases (such as other endopeptidases) are absent or present in only small amounts.

[0079] Therefore, in a preferred embodiment of the method of the present invention, the microbial serine protease accounts for at least 80%, preferably at least 90%, and more preferably at least 95% (w / w enzyme protein) of the exogenous protease used in the method.

[0080] In another preferred embodiment of the method of the present invention, the serine protease preparation is the only exogenous protease used in the method.

[0081] Example Example 1: High-throughput micro-yogurt fermentation using different starter cultures and endopeptidase dosages Miniature Yogurt Preparation: Two pasteurized and homogenized milks [(1) 10% protein: 1% fat, 10% protein, containing skim milk, cream, whey protein concentrate (WPC) 550 (commercially available from Fonterra) and milk protein concentrate (MPC) 852A, or (2) 4.5% protein: 1% fat, 4.5% protein, containing skim milk and cream] were supplemented with 5% blue-green pH indicator (1 g bromocresol green, 1 g bromocresol purple, 2 mL 4 M NaOH, diluted to 1 L with MilliQ water). 200 µl of each milk was placed in 1.5 mL Fliptube (Hamilton) at 43°C with a starter culture (commercially available from Chr. Hansen) and different doses of food-grade microbial endopeptidase Galaya. ® Fermentation was carried out using Smooth (commercially available from Novozymes) until pH 4.6 was reached, followed by storage at 4°C. The starter culture used in this experiment was selected based on different strain compositions and contained *Lactobacillus delbrueckii*, *Streptococcus thermophilus*, and *Lactobacillus rhamnosus*. Fermentation was performed in quadruplicate.

[0082] Table 1: Composition of milk matrix, protein supplementation source, starter culture, and dosage of microbial endopeptidase used in micro yogurt fermentation experiments.

[0083] The time points for reaching pH 4.6 and transferring each fermentation sample were determined by placing 96-well plates containing Fliptube fermentation replication on a benchtop scanner and scanning the color value of each well every 4 minutes as a proxy for pH changes over time (Poulsen, VK. et al. "High-throughput screening for texturing"). Lactococcus [High-throughput screening for textured Lactococcus strains] FEMS Microbiology Letters 366.2 (2019): fnz001. Once fermentation reached pH 4.6, the corresponding Fliptube was automatically transferred from 43°C to 5°C by a Hamilton robot. For samples that had not reached pH 4.6 after 20 h, they were transferred to 5°C. All samples were stored at 5°C until PCA extraction was performed the following day.

[0084] analyze: Fermentation samples and controls were homogenized in a Fliptube with manual stirring, followed by the addition of 1 mL of PCA solution to each sample. The samples were then vortexed and rotated on the rotor for 30 min. Subsequently, the samples were centrifuged at 15000 g for 10 min at 4 °C, and 800 µl of the supernatant was transferred to a 96-well plate for further LC-MS analysis. For LC-MS, the analytes were derivatized using an AccQ-Tag Ultra and analyzed on a Waters I-Class UHPLC system equipped with a Waters Xevo TQ-XS triple quadrupole mass spectrometer (using positive electrospray ionization and internal standard).

[0085] result: The addition of serine protease increased the concentration of free amino acids in all samples, with a stronger effect in 10% yogurt than in 4.5% yogurt. In 10% yogurt, the levels ranged from 29 to 86 mg / 100 mL, compared to 21 to 26 mg / 100 mL in 4.5% yogurt. Figure 1 A similar trend was observed with free essential amino acids, with increases ranging from 79 to 207 mg / 100 mL in 10% yogurt, compared to smaller increases in 4.5% yogurt. Figure 2 Among the essential amino acids, histidine, isoleucine, leucine, lysine, methionine, and phenylalanine showed the greatest increases, with isoleucine, leucine, and lysine increasing by as much as 2, 3–10, and 10–20 mg / 100 mL, respectively. Figure 3 Importantly, the increase in total free amino acids and essential free amino acids mediated by serine proteases was consistent across all tested starter cultures.

[0086] Example 2: Effects of endopeptidase addition on degree of hydrolysis and peptide formation during fermentation and simulated digestion Experimental plan: This experiment investigated the effects of adding endopeptidase on protein hydrolysis and peptide formation during lactic acid bacteria fermentation and during simulated gastrointestinal digestion of the produced yogurt. The experiment was conducted under enzyme-free control conditions and with endopeptidase-treated samples (low, medium, and high dose levels).

[0087] The effect of endopeptidase addition was evaluated by determining the degree of hydrolysis (DH%), peptide number, peptide abundance, and putative formation of functional peptides during the neutralization and digestion of fermented yogurt. Simulated gastrointestinal digestion was performed according to the INFOGEST static in vitro digestion protocol described by Brodkorb et al. (2019), and samples were collected after incubation for 0, 15, 30, 60, and 120 min during the intestinal phase.

[0088] Fermentation agent: Yogurt starter (YoFlex) ® The starter culture YF-L904, which is commercially available from Chr. Hansen and contains Streptococcus thermophilus and Lactobacillus delbrueckii subsp. bulgaricus, can be used.

[0089] Endopeptidase preparations: Using food-grade microbial endopeptidase Galaya ® Smooth formulation. The enzyme is added to the emulsion substrate immediately before inoculation with the starter culture.

[0090] Three enzyme dosage levels were tested: • Low dose: corresponds to an activity of 36 KPRU / kg protein. • Medium dose: corresponds to an activity of 72 KPRU / kg protein. • High dose: corresponds to an activity of 96 KPRU / kg protein.

[0091] emulsion base: The emulsion base is formulated as described in Table 2.

[0092] Table 2: Composition of the milk matrix and sources of protein supplementation used in the yogurt fermentation experiment.

[0093] Testing the production process of the product: The milk base was mixed and rehydrated at 60°C for one hour, then homogenized at 65°C and 130 / 40 bar, and heat-treated at 95°C for 5 minutes via a heat exchanger, followed by cooling to 15°C. Microbial endopeptidase was then added at the desired dosage (0, 36, 76, or 96 U / kg protein), and yogurt starter (YoFlex) was added at a dosage of 500 U / 2500 L. ® F-DVS YF-L904 was inoculated into the milk-based medium. Fermentation was carried out at 43°C until pH 4.6 was reached.

[0094] analyze: • Hydrolysis degree analysis of OPA: The degree of hydrolysis (DH) was determined using the standard orthophthalaldehyde (OPA) assay. OPA reacts with free primary amino groups in the presence of DTT, and the resulting product can be measured spectrophotometrically at 340 nm. For the assay, the sample was diluted with Milli-Q water to a protein concentration of approximately 0.05%–0.25%, and a two-fold serial dilution was prepared. A total of 20 µL of diluted sample was then transferred to a microplate, followed by the addition of 200 µL of OPA reagent. After shaking the plate, the absorbance was measured immediately at A340 nm. Quantification was performed using an L-serine standard curve, and DH was calculated relative to the protein content of the sample. OPA reagent was freshly prepared daily and stored protected from light. Briefly, 10 mL of 0.15 M sodium carbonate stock solution and 10 mL of 0.6 M sodium bicarbonate stock solution were combined, followed by the addition of 88 mg DTT, 80 mg OPA (dissolved in 2 mL ethanol), and 1 mL of 10% SDS. Dilute the solution to 100 mL with Milli-Q water and store away from light until use.

[0095] • Peptide sequencing via LC-MS: For peptide sequencing, the sample was first adjusted to the desired protein concentration, then reduced with DTT, alkylated with iodoacetamide (IAA), and precipitated with trichloroacetic acid (TCA). Specifically, 40 µL of sample was combined with 5 µL of 0.5 M DTT and incubated at 95 °C and 650 rpm for 10 min. Then, 5 µL of 0.5 M IAA was added, and the sample was incubated in the dark for 20 min. Next, 20 µL of 50% TCA was added to achieve a final concentration of approximately 10%–15% TCA, and the sample was incubated overnight at -20 °C. After thawing, the sample was centrifuged at 3,500 × g for 45 min at 4 °C, and the supernatant was transferred to a new tube.

[0096] For LC-MS analysis, samples were loaded onto Evotip pipettes according to the manufacturer's instructions, using 4 µL of sample per tip, followed by 20 µL of solvent A. Samples were analyzed using a gradient of 200 samples / day on a timsTOF HT, with an acquisition time of approximately 5.5 min per sample. An optimized MS acquisition method was applied, as described by Thesbjerg et al. (2024), enabling MS stepping to allow reacquisition of the target after 0.1 min, and selecting ions with charges of 1–6 for MS / MS. Raw data were searched against the bovine Swiss-Prot proteome database in PEAKS using a non-enzymatic search strategy, allowing peptides of 6–45 amino acids and a maximum of four PTMs per peptide. Fixed and variable modifications include carboxylation (Cys, fixed) and oxidation (Met, variable), as well as additional variable PTMs, including carboxylation (C, H, K, E, and N-terminals), phosphorylation (STY), lactosylation (K), and pyroglutamylation (Q / E).

[0097] • Peptide size distribution via SEC-HPLC-UV Peptide fingerprinting and size distribution were analyzed by SEC-HPLC-UV. Samples were pre-diluted to 2 mg / mL protein, centrifuged at 8,000 × g for 10 min at 4 °C, filtered through a 0.22 µm PTFE membrane, and then injected. Analysis was performed using a Bio SEC-5 column (7.8 × 300 mm, 150 Å, 5 µm) and a guard column, with 150 mM sodium phosphate buffer (pH 7.0) as the mobile phase. The flow rate was 1 mL / min, the injection volume was 10 µL, and detection was performed at 214 nm and 280 nm. Protein / peptide standards were used to analyze the resulting peptide range.

[0098] result: Compared to the untreated control, the addition of serine protease increased the relative degree of hydrolysis (DH%) in both high-protein yogurt formulations. In the 8% protein yogurt, DH increased to 125% and 126% at 36 and 72 kPRU / kg, respectively, while it increased to 113% at 96 kPRU / kg. In the 12% protein yogurt, the increase was smaller, with DH values ​​of 110%, 104%, and 107% at 36, 72, and 96 kPRU / kg, respectively. Overall, these data suggest that the addition of serine protease promotes higher degrees of hydrolysis in both yogurt formulations, with the strongest effect observed in the 8% formulation. Figure 4 ).

[0099] Based on the hydrolysis degree data, further analysis was conducted to assess whether serine protease treatment altered peptide length distribution and peptide abundance. For example... Figure 5 As shown, enzyme addition increased peptide formation in both high-protein yogurt formulations. In 8% protein yogurt ( Figure 5 (a and b) All tested doses increased both the number and relative abundance of detected peptides, with the strongest enrichment observed in the 6–25 amino acid range. The peptides with the largest increases observed were approximately 8, 12, and 21–22 amino acids, indicating a shift in the peptide profile towards lower molecular weights. A similar pattern was observed in 12% protein yogurt. Figure 5 (c) and (d), where enzyme addition primarily increased peptide number and abundance within the same 6–25 amino acid range. In summary, these data indicate that the addition of serine proteases promoted proteolysis and the accumulation of smaller peptides in both yogurt matrices.

[0100] To assess whether changes in protein hydrolysis in the yogurt matrix persisted under simulated gastrointestinal conditions, the samples were analyzed using the INFUGEST digestion protocol. Figure 6 As shown, at the onset of the intestinal phase, 8% protein yogurt treated with microbial endopeptidase exhibited increased peptide number and peptide strength at all tested doses, particularly in the 6–15 amino acid range. Figure 6 a and b). In 12% protein yogurt, 36 KPRU / kg increased peptide abundance within the same size range, while 76 and 96 KPRU / kg did not show a significant increase relative to the control at that time point. Figure 6 (c and d). Overall, these data indicate that the addition of serine proteases enhances the production of small peptides during simulated digestion, with a stronger effect observed in formulations containing 8% total protein.

[0101] based on Figure 6 The digestion data were used to examine the release of the desired peptide NAVITPTL, which was previously thought to support bone formation and maintain bone strength. Specifically, Karvande et al. and Reddi et al. demonstrated its effect on osteoblasts in vitro, including increasing markers associated with matrix formation and mineralization; and in a rat model of bone loss, administration of NAVITPTL improved bone mineral density and bone microstructure parameters compared to controls.

[0102] like Figure 7 As shown, at the onset of the intestinal phase, the relative abundance of NAVPITPTL in 8% protein yogurt at all tested doses showed a significant increase after the addition of serine protease. Figure 7 (Left side image). In 12% protein yogurt ( Figure 7(See right subplot). Treatments of 36 and 76 kPRU / kg resulted in only modest increases relative to the control, while 96 kPRU / kg significantly increased peptide abundance. Overall, these data indicate that the enzyme promotes NAVIPTL production during digestion, with the strongest effect observed at the highest dose in the 12% formulation.

[0103] To further characterize the protein hydrolysis changes observed by LC-MS, size exclusion HPLC was used to analyze yogurt samples. Figure 8 As shown, the addition of serine proteases increased the abundance of protein fragments and larger peptides (6.7–45 kDa) in both formulations. In the 8% protein yogurt, the signal increased from 2 mAU in the control to 19 mAU at 36 kPRU / kg, and 23.3 mAU at 76 and 96 kPRU / kg, representing increases of 9.5-fold and 11.7-fold, respectively. In the 12% protein yogurt, the signal increased from 15 mAU to approximately 26 mAU at all enzyme doses, representing an increase of 1.7-fold. A similar dose-dependent pattern was observed for the 1.7–6.7 kDa peptide fraction. In the 8% yogurt, the signal increased from 4.6 mAU to 10, 15, and 16.2 mAU at 36, 76, and 96 kPRU / kg, respectively, representing increases of 2.2-fold, 3.3-fold, and 3.5-fold, respectively. In 12% yogurt, the signal increased from 3.7 mAU to 12, 14.5, and 17 mAU, representing increases of 3.2-fold, 3.9-fold, and 4.6-fold, respectively. Overall, size exclusion HPLC showed that enzyme treatment shifted the protein profile towards higher abundance fragmented low molecular weight substances in a dose-dependent manner.

[0104] To further evaluate the role of serine proteases in the formation of very small peptides during digestion, peptides with fewer than 7 amino acids during the intestinal phase of the INFOGEST protocol were analyzed. For example... Figure 9 and Figure 10 As shown, in 8% protein yogurt, the relative abundance of these peptides showed a significant dose-dependent increase throughout digestion, with increases ranging from 106% to 157% relative to the enzyme-free control. This effect was evident at the onset of the intestinal phase and remained elevated over time, with the strongest signal at higher enzyme doses. In 12% protein yogurt ( Figure 10The response was not uniform, with increases ranging from 106% to 162% throughout digestion. Specifically, the 36 KPRU / kg treatment increased peptide abundance in the early intestinal phase, while the 76 and 96 KPRU / kg treatments showed weaker initial effects, becoming more significant at later time points (particularly 60 and 120 min). Overall, these data suggest that the addition of serine proteases promotes the formation of very small peptides during simulated intestinal digestion, with a stronger and more consistent effect observed in the 8% formulation.

[0105] In summary, these results indicate that the addition of microbial endopeptidases produces a more extensively pre-hydrolyzed yogurt protein matrix, evident in both products and showing increased degree of hydrolysis, altered peptide profiles, and increased generation of medium to low molecular weight peptides, protein fragments, and very small peptides during simulated digestion. This shift in proteolytic activity suggests a more readily digestible protein system with potentially faster gastrointestinal breakdown and earlier peptide release and absorption. The increased release of candidate bioactive peptides further supports the potential relevance of this approach to human health.

[0106] Example 3: Effects of endopeptidase addition on viscosity and sensory properties during fermentation Experimental plan: The purpose of this experiment is to study the effect of adding endopeptidase on the viscosity and sensory properties of fermented dairy products.

[0107] The following parameters were evaluated: ■ Endopeptidase addition: No enzyme (control) and with endopeptidase. ■ Endopeptidase dosage levels: Low, medium, and high doses were tested.

[0108] Fermentation products were compared based on shear stress and sensory properties (including bitterness).

[0109] Fermentation agent: Yogurt starter (YoFlex) ® The starter culture YF-L904, which is commercially available from Chr. Hansen in Denmark, contains Streptococcus thermophilus and Lactobacillus delbrueckii subsp. bulgaricus.

[0110] Endopeptidase preparations: Using food-grade microbial endopeptidase Galaya ® Smooth formulation. The enzyme is added to the emulsion substrate immediately before inoculation with the starter culture.

[0111] Two enzyme dosage levels were tested: • Low dose: corresponds to an activity of 36 KPRU / kg protein. • Medium dose: corresponds to an activity of 72 KPRU / kg protein. • High dose: corresponds to an activity of 96 KPRU / kg protein.

[0112] emulsion base: Composition of emulsion substrate The emulsion base is prepared as follows:

[0113] analyze • Viscosity Viscosity was measured on an Anton Paar Modular Compact Rheometer 302e (using a CC27 bob cup set-up). Flow sweeps were used to generate different shear rates. All measurements were performed at 13°C.

[0114] • Sensory evaluation The sensory characteristics of fermented dairy products (including bitterness, sourness, mouthfeel, and astringency) were evaluated.

[0115] result: The addition of microbial endopeptidase significantly reduced the viscosity-related shear stress in both 8% and 12% protein emulsions. In both matrices, the enzyme-free control showed the highest shear stress, while the enzyme-treated samples exhibited significantly lower shear stress values. Specifically, at 8% protein, the addition of endopeptidase significantly reduced shear stress, with a substantial reduction observed at 36 U / kg and a further reduction at 76 KPRU / kg. At 96 KPRU / kg, the shear stress remained at a similarly low level. A similar effect was observed at 12% protein, where enzyme treatment also caused a significant reduction in shear stress compared to the reference. Figure 11 ).

[0116] Sensory evaluation showed that the addition of microbial endopeptidase did not increase bitterness in fermented dairy products with 8% or 12% protein. Figure 12 Simultaneously, enzyme treatment also affected other sensory properties in both protein matrices, particularly mouthfeel. These results indicate that endopeptidases can alter desired texture-related sensory properties without increasing bitterness. Figure 12 ).

[0117] References 1. Norton, L. E.&Layman, D. K. Leucine regulates translationinitiation of protein synthesis in skeletal muscle after exercise. J. Nutr. 136, 533S–537S (2006). 2. Wolfe, R. R. Branched-chain amino acids and muscle proteinsynthesis in humans: myth or reality? J. Int. Soc. Sports Nutr. 14, 30 (2017). 3. Churchward-Venne, T. A., Burd, N. A.&Phillips, S. M. Nutritionalregulation of muscle protein synthesis with resistance exercise. J. Appl. Physiol. 113, 155–167 (2012). 4. Boirie, Y. et al. Slow and fast dietary proteins differentlymodulate postprandial protein accretion. Proc. Natl Acad. Sci. USA 94, 14930–14935 (1997). 5. Dangin, M. et al. The digestion rate of protein is an independentregulating factor of postprandial protein retention. Am. J. Physiol. Endocrinol. Metab. 280, E340–E348 (2001). 6. Calbet, J. A. L.&Holst, J. J. Gastric emptying, gastric secretionand enterogastrone response after administration of milk proteins or peptidesin humans. Eur. J. Clin. Invest. 34, 494–500 (2004). 7. Tang, J. E. et al. Ingestion of whey hydrolysate, casein, or soyprotein isolate: effects on mixed muscle protein synthesis after resistanceexercise in young men. J. Appl. Physiol. 107, 987–992 (2009). 8. Daniel, H. Molecular and integrative physiology of intestinalpeptide transport. Annu. Rev. Physiol. 66, 361–384 (2004). 9. Brandsch, M., Knütter, I.&Bosse-Doenecke, E. Pharmacological andphysiological substrates of the peptide transporter PEPT1. J. Pharm. Pharmacol. 60, 543–585 (2008). 10. Rubio-Aliaga, I.&Daniel, H. Peptide transporters and their rolesin physiological processes and drug disposition. Xenobiotica 38, 1022–1042(2008)。

Claims

1. Galaya ® Smooth is used to improve the digestibility of protein in fermented dairy products.

2. The use according to claim 1, wherein the fermented dairy product is drinkable yogurt.

3. The use according to claim 2, wherein the protein content of the drinkable yogurt is in the range of 4.5 wt%-12 wt%, preferably 8 wt%-12 wt%.

4. The use according to claim 2 or 3, wherein Galaya ® This use of Smooth also increases the concentration of free amino acids in the drinkable yogurt.

5. The use according to claim 4, wherein the concentration of the free amino acid is increased by 50 wt%-120 wt%, preferably 70 wt%-120 wt%.

6. The use according to claim 4 or 5, wherein the increase in the concentration of free amino acids shall include an increase in the concentration of free essential amino acids, for example, in a drinkable yogurt with a protein content of 10 wt%, the level of free essential amino acids is increased to 79 to 207 mg / 100 mL of drinkable yogurt; or, for example, in a drinkable yogurt with a protein content of 4.5%, the level of free essential amino acids is increased to 48 to 69 mg / 100 mL of drinkable yogurt.

7. The use according to claim 6, wherein the increase of the free essential amino acid includes an increase in the concentration of free lysine, preferably an increase in the concentration of free lysine to 10-20 mg / 100 mL.

8. The use according to claim 6 or 7, wherein the increase of the free essential amino acid includes an increase in the concentration of free leucine, preferably an increase in the concentration of free leucine to 2-10 mg / 100 mL.

9. The use according to any one of claims 6, 7 or 8, wherein the increase of the free essential amino acid includes an increase in the concentration of free isoleucine, preferably an increase in the concentration of free isoleucine to about 2 mg / 100 mL.

10. The use according to any one of claims 2-9, wherein the Galaya ® The use of smoothing also increases the degree of hydrolysis of the drinkable yogurt by 4%-26%.

11. The use according to any one of claims 2-10, wherein during in vitro digestion according to the INFUGEST protocol, the Galaya, relative to the enzyme-free control, ® The use of Smooth also increases the concentration of peptides with fewer than 7 amino acids in the drinkable yogurt, increasing the concentration by 6%–57% in drinkable yogurt with a protein content of 8 wt% and by 6%–62% in yogurt with a protein content of 12 wt%.

12. Galaya ® Smooth is used to increase the concentration of a desired peptide in drinkable yogurt, wherein the desired peptide is NAVPITPTL, preferably wherein, during in vitro digestion according to the INFOGEST protocol, the concentration of the desired peptide is increased by 144%–191% in 8% protein yogurt and by 13%–255% in 12% protein yogurt, relative to an enzyme-free control.

13. Galaya ® Smooth is used to improve texture-related sensory properties, preferably a thick, viscous and / or smooth feel in the mouth, without increasing bitterness.

14. A method for preparing a fermented dairy product containing a protein with improved digestibility, the method comprising using Galaya ® Smooth-treated milk base, wherein the protein content of the milk base is in the range of 4.5 wt%–12 wt%, preferably 8 wt%–12 wt%.

15. The method of claim 14, wherein the dairy product is drinkable yogurt.

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