US 201503,42236A1 (19) United States (12) Patent Application Publication (10) Pub. No.: US 2015/0342236A1 BHOWMK et al. (43) Pub. Date: Dec. 3, 2015

(54) ENZYMATIC PROCESS (60) Provisional application No. 61/074,167, filed on Jun. 20, 2008. (71) Applicant: Givaudan SA, Vernier (CH) Publication Classification (72) Inventors: Tarun BHOWMIK, Mason, OH (US); Stefka Ivanova MYAKA, Loveland, OH (51) Int. Cl. (US); Johan Peter VAN LEERSUM, A2.3L I/237 (2006.01) Morrow, OH (US); Roy Wade SMITH, A2.3L I/23 (2006.01) Cold Spring, KY (US) (52) U.S. Cl. CPC ...... A23L I/2375 (2013.01); A23L I/23 (21) Appl. No.: 14/824,682 (2013.01); A23 V 2002/00 (2013.01) (22) Filed: Aug. 12, 2015 (57) ABSTRACT O O Provided is an enzymatic process that hydrolyzes spinach Related U.S. Application Data plant material to form a salt-enhancing ingredient, the formed (63) Continuation of application No. 12/996,731, filed on salt-enhancing ingredient, food products comprising said Dec. 7, 2010, filed as application No. PCT/CH2009/ salt-enhancing ingredient and a method of enhancing the 000201 on Jun. 15, 2009. salty taste of food products. US 2015/0342236 A1 Dec. 3, 2015

ENZYMATIC PROCESS 0016 (8) The process as described under item (7) wherein the Lactobacillus microorganism is selected from the TECHNICAL FIELD group consisting of L. plantarum, L. casei, L. brevis and L. 0001 Disclosed is a novel ingredient and the enzymatic helveticus. process to form said ingredient to enhances saltiness in food 0017 (9) The salt-enhancing ingredient formed by the products, in particular in food products with a low or reduced process as described under any one of items (1) to (8). Sodium content, to improve their taste. 0018 (10) The salt-enhancing ingredient as described under item (9) which is concentrated at least 1.5 times by BACKGROUND removing water. 0019 (11) The salt-enhancing ingredient as described 0002. A high amount of sodium intake is considered to be under any one of items (9) to (10) wherein the salt-enhanc detrimental to health and therefore there is a desire to reduce ing ingredient is spray-dried. the amount of sodium chloride (NaCl) in food products, with 0020 (12) A flavor composition for food products com out reducing the desired salty taste at the same time. The salty prising the salt-enhancing ingredient as described under taste is very important to the perceived flavour intensity and any one of items (9) to (11) and one or more food-grade profile, especially for savory food products. excipient. 0003. There exists a need in the food industry to provide ingredients that enhance the salty taste of food products so 0021 (13) The flavor composition as described under item that sodium/NaCl can be reduced. (12) wherein the concentration of the salt-enhancing ingre 0004 Potassium Chloride (KCl) is used to replace other dient of claim 9 is 0.25 to 400 ppm based on the use of the salts, particularly NaCl. If KCl is used in the desired concen unconcentrated Salt-enhancing ingredient. Useful concen tration to reduce NaCl, an undesirable bitter and metallic taste trations for the salt-enhancing ingredient (unconcentrated) is perceived by the consumer. Furthermore, certain individu in flavor compositions or food products include 0.25 to 300 als desire to avoid KCl because of health concerns. It would ppm, 0.25 to 200 ppm, 0.5 to 100 ppm, and 0.5 to 20 ppm. therefore be of interest to find a product that is able to enhance 0022 (14) A food product comprising the salt-enhancing the salty taste of NaCl so that KCl can be partially or com ingredient as described under any one of items (9) to (11). pletely replaced. 0023 (15) The food product as described under item (14) 0005 Spinach or its hydrolysates have not been known for wherein the concentration of the salt-enhancin ingredient a salt-enhancing effect. as described under any one of items (9) to (11) is 0.25 to 400 ppm based on the use of the unconcentrated salt SUMMARY enhancing ingredient. 0024 (16) The food product as described under any one of 0006 Various aspects of the invention comprise the fol items (14) to (15) which is a reduced or low sodium food lowing: product. 0007 (1) A process of forming a salt-enhancing ingredient (0025 (17) The food product as described under item (16) comprising the steps of wherein the sodium chloride concentration is 0.15% (wit/ 0008 (i) forming an aqueous slurry of spinach plant mate wt) to 3% (wit/wt). rial, and 0026 (18) The food product as described under item (16) 0009 (ii) forming a hydrolysate of spinach plant material wherein the sodium chloride concentration is 0.15% (wit/ by Subjecting it to an enzymatic hydrolysis using one or wt) to 1.5% (wit/wt). more proteolytic enzymes. 0027 (19) The reduced or low sodium food product as 0010 (2) The process as described under item (1), wherein described under any one of items (16) to the formed salt-enhancing ingredient is inactivated by 0028 (18) additionally comprising KC1, optionally in a heating. concentration of 0.1% to 2% (wit/wt) KC1. 0011 (3) The process as described under any one of items 0029 (20) A method of providing a food product (1) to (2) wherein the one or more proteolytic enzymes are enhanced in saltiness wherein the salt-enhancing ingredi Selected from the group consisting of proteinase, pepti ent as defined under any one of items (9) to (11) is admixed dase, and glutaminase. to a food product. 0012 (4) The process as described under any one of items 0030 (21) The method as described under item (16) (1) to (3) wherein the one or more proteolytic enzymes wherein the food product is a reduced or low sodium food comprise both proteinase and peptidase enzymes. product optionally containing KC1, optionally in a concen 0013 (5) The process as described under any one of items (1) to (4) wherein the one or more proteolytic enzymes tration of 0.1% to 2% (wit/wt) KC1. comprise an enzyme preparation from Aspergillus Oryzae (UmamizymeTM) and the hydrolysis is performed at 40°C. DETAILED DESCRIPTION to 60° C. 0031 Surprisingly, it has been found that when spinach is 0014 (6) The process as described under any one of items treated enzymatically with one or more proteolytic enzymes (1) to (5) wherein the hydrolysate is formed by subjecting including, without limitation, the enzyme classes of protease, the spinach plant material to an enzymatic hydrolysis using peptidase, and glutaminase, an ingredient can be formed that one or more carbohydrase enzymes in parallel or Subse has an enhancing effect on the perception of salty taste in food quent to enzymatic hydrolysis by the one or more pro products and exhibits a salty taste of higher intensity. teolytic enzymes. 0032. By salt-enhancing is meant the effect of an ingredi 00.15 (7) The process as described under any one of items ent on the salty taste in food which is found more pronounced (1) to (6) wherein the hydrolysate is subjected to fermen (stronger, enhanced) in its taste intensity and/or longer in its tation using a Lactobacillus. duration as analyzed by trained panellists sensitive to salty US 2015/0342236 A1 Dec. 3, 2015 taste, when comparing food comprising an ingredient with a efficiently break down a protein both from within and from salt-enhancing effect to food without an added salt-enhancing the ends of each protein and resulting peptide. ingredient. 0041) Useful proteolytic enzymes include, without limita 0033. The enhanced intensity of the perception of salty tion, an enzyme with one or more of the following activities: taste by the salt-enhancing ingredient can be increased by protease, peptidase, glutaminase (including, without limita additionally using a carbohydrase enzyme, either in parallel tion, L-glutamine-amido-hydrolase (EC 3.5.1.2)), endopro or consecutively, in its formation. tease, serine endopeptidase, Subtilisin peptidase (EC 3.4.21. 0034) To avoid off-tastes that may become noticeable in 62). Some food products when using higher concentrations of the 0042. Other proteolytic enzymes are useful as well, and a salt-enhancing ingredient (a higher concentration may be, for great variety is known and available; some additional types example, about 50 ppm or more, about 100 ppm or more, and examples are given below. about 200 ppm or more, or about 300 ppm or more), it is 0043 Proteolytic enzymes (also called proteases, protein advantageous to include glutaminase in the enzymatic treat ases, or peptidases) are currently classified in six groups ment. including serine protease, threonine protease, cysteine pro 0035. The enhanced intensity of the perception of salty tease, aspartic acid protease, metalloprotease, and glutamic taste can be further increased by an optional fermentation step acid protease. Proteolytic enzymes can cut at the end of a employing Lactobacillus bacteria, for example, Lactobacil protein (exopeptidases) or attack internal peptide bonds of a lus plantarum. protein (endopeptidases). Exopeptidases include, without limitation, aminopeptidases, carboxypeptidases, and carbox Spinach ypeptidase A. Endopeptidases include, without limitation, trypsin, chymotrypsin, pepsin, papain, and elastase. 0036 “Spinach' as used herein refers to the green leaves 0044 Proteolytic enzymes (EC 3.4 and EC 3.5) are clas and/or stalks of a flowering plant in the family of Amaran sified by an EC number (enzyme commission number), each thaceae (formerly known as Chenopodiaceae), including class comprises various known enzymes of a certain reaction Spinacia oleracea, and the closely related species Beta vul type. garis (also known as chard, spinach beet, silverbeet or per 0045 EC 3.4 comprises enzymes acting on peptide bonds petual spinach); Atriplex spec., including A. hortensis (also (peptidases/proteinases) and EC 3.5 comprises enzymes that known as orache, French spinach, or mountain spinach); and act on carbon-nitrogen bonds other than peptide bonds. Chenopodium spec., including C. bonus-henricus and C. 0046 Examples for EC 3.4 include, without limitation, the album (also known as Wild spinach, Fathen, Good King following: aminopeptidase (EC 3.4.11), dipeptidase (3.4.13), Henry or Lincolnshire spinach). dipeptidyl-peptidase (3.4.14), peptidyl-dipeptidase (3.4.15), 0037 Spinach includes three to four types, Savoy, Flat/ serine-carboxypeptidase (3.4.16), metallocarboxypeptidase Smooth leaf spinach, Semi-savoy, and Baby. (3.4.17), cysteine-carboxypeptidase (3.4.18), omegapepti 0038 Savoy has dark green, crinkly and curly leaves. The dase (3.4.19), serine-endopeptidase (3.4.21), cysteine-en Bloomsdale and the Tyee are popular Savoy type varieties. dopeptidase (3.4.22), aspartate-endopeptidase (3.4.23), met Flat/smooth leaf spinach has broad smooth leaves that are alloendopeptidase (3.4.24), threonine-endopeptidase easier to clean and is grown for canned, frozen or processed (3.4.25). foods. Semi-savoy is a hybrid variety with slightly crinkled 0047. Examples for EC 3.5 include, without limitation, leaves. Semi-savoy includes Five Star, a widely grown vari proteolytic enzymes that cleave in linear amides (3.5.1), for ety. Baby is of the flat-leaf type and its leaves are usually no example, without limitation, glutaminase (EC 3.5.1.2). longer than three inches. Examples of spinach varieties 0048 Various proteolytic enzymes are commercially include, without limitation, America, Bloomsdale or Blooms available; the following proteolytic enzymes are available dale Long Standing, Dominant, Giant Winter, Horenso, from Sigma-Aldrich: Achromopeptidase, Aminopeptidase, Medania, Sigmaleaf, Space, Trinidad, Tyee (Savoy type), Ancrod, Angiotensin Converting Enzyme, Bromelain, Olympia, Melody, Winter Bloomsdale, Bordeaux, Koto, Calpain, Calpain I, Calpain II, Carboxypeptidase A, Carbox Lazio, Vienna, Marathon, Seven R. Baker, Cascade, Olympia, ypeptidase B, Carboxypeptidase G. Carboxypeptidase P. Polka, Rainier, Shasta, Wolter, Bossanova, Bolero, Coho, Carboxypeptidase W. Carboxypeptidase Y. Caspase, Caspase Ambassador, Rainier, Rhythm 9, Hybrid #7, Skookum, Bejo 1. Caspase 2, Caspase 3, Caspase 4, Caspase 5, Caspase 6. 1369, Splendor, Indian Summer, Avon, Correnta, Nordic IV. Caspase 7.Caspase 8, Caspase 9, Caspase 10, Caspase 13, Savoy Supreme, Space, Spokane, Springfield, Steadfast, Uni Cathepsin B, Cathepsin C, Cathepsin D. Cathepsin G. Cathe pak 12, Mazurka, Chinook II, Hybrid 424, St. Helens, Baker, psin H. Cathepsin L. Imperial Express and Imperial Star. 0049 Chymopapain, Chymase, Chymotrypsin, a-Clostri pain, Collagenase, Complement Clr, Complement Cls, Enzymes Complement Factor D. Complement factor I, Cucumisin, Dipeptidyl Peptidase IV, Elastase, leukocyte, Elastase, pan 0039 Exemplary useful enzyme classes include pro creatic, Endoproteinase Arg-C, Endoproteinase Asp-N. teolytic enzymes that hydrolyze bonds in a protein, and Endoproteinase Glu-C, Endoproteinase Lys-C. Enterokinase, optionally, a carbohydrase. Factor Xa, Ficin, Furin, Granzyme A, Granzyme B, HIV 0040 Proteolytic enzyme preparations usually contain Protease, IGase, Kallikrein tissue, Leucine Aminopeptidase proteinases, which hydrolyze proteins to form Small peptides, (General), Leucine aminopeptidase, cytosol, Leucine ami and peptidases, which hydrolyze Small proteins or peptides, nopeptidase, microsomal, Matrix metalloprotease, Methion usually to release amino acids from their terminal ends. Often ine Aminopeptidase, Neutrase, Papain, Pepsin, Plasmin, Pro proteinases and peptidases with both endopeptidase and lidase, Pronase E, Prostate Specific Antigen, Protease, exopeptidase activity are included in Such preparations, to Alkalophilic from Streptomyces griseus, Protease from US 2015/0342236 A1 Dec. 3, 2015

Aspergillus, Protease from Aspergillus Saitoi, Protease from izymeTM, a protease/peptidase (Amano, Nagoya, Japan), or Aspergillus sojae, Protease (B. licheniformis) (Alkaline), Flavorpro 373TM, a subtilisin peptidase (Biocatalysts, Protease (B. licheniformis) (Alcalase), Protease from Bacil Cardiff, UK), may be used. lus polymyxa, Protease from Bacillus sp., Protease from Bacil 0057 All enzymes used should be food-grade. lus sp (Esperase), Protease from Rhizopus sp., Protease S, Proteasomes, Proteinase from Aspergillus Oryzae, Proteinase Enzymatic Hydrolysis 3, Proteinase A, Proteinase K. Protein C, Pyroglutamate ami 0.058 Enzymatic hydrolysis is performed under condi nopeptidase, Renin, Rennin, Streptokinase, Subtilisin, Ther tions suitable for all enzymes employed. As will be apparent molysin, Thrombin, Tissue Plasminogen Activator, Trypsin, to the skilled person, the temperature and pH should be within Tryptase, Urokinase. a suitable range for hydrolysis to occur to the desired degree. 0050. One or more of the proteolytic enzymes described The incubation length will vary accordingly, with shorter herein can be combined with a carbohydrase to increase the incubations when conditions are nearer to the optimum con salt impact of the formed Salt-enhancing ingredient. ditions. Usually 1 to 48 hours will be sufficient, for example, 0051. Useful enzyme combinations include, without limi 10 to 24 hours. Necessary ions, if required or beneficial for the tation, combinations wherein at least one proteolytic enzyme chosen enzyme(s), should be present, as the skilled 10 person is combined with at least one carbohydrase. will be aware. Stirring the incubation mix, for example 50 to 0052. Useful carbohydrase enzymes to break down carbo 500 rpm, or 100 to 200 rpm, usually improves the hydrolysis. hydrate plant material include, without limitation, carbohy Some enzymes tolerate stirring better than others. Tolerance drases with one or more of the following activities: beta towards one factor often depends on the other factors. Such glucanase (including, without limitation, 1.3-beta-glucan information on suitable conditions is readily available for gluco-hydrolase (EC 3.2.1.58)), beta-amylase, cellulase, many enzymes and otherwise can be easily determined. hemicellulase, Xylanase. 0059 A number of enzyme preparations, including Cera 0053 For example, the following combinations are non mixTM, AlcalaseTM, ViscozymeTM, and UmamizymeTM, will limiting examples of useful enzymes: work well in a liquified slurry of spinach in water at a tem perature from 40° C. to 55° C., for example about 45° C. to about 55°C., without pH adjustment or any added co-factors. Protease? Peptidase, Glutaminase Carbohydrase Others may need or will benefit from pH or temperature Protease beta-glucanase adjustment, or additives. UmamizymeTM will tolerate tem Protease beta-amylase peratures from about 40°C. to about 60°C., with an optimum Protease cellulase at around 55°C. UmamizymeTM originates from Aspergillus Protease hemicellulase Oryzae and is rich in endopeptidase and exopeptidase activity. Protease Xylanase Endoprotease beta-glucanase 0060 Sufficient units of the enzyme to achieve a substan Endoprotease beta-amylase tial hydrolysis until the desired degree of salty taste is Endoprotease cellulase achieved should be used. Endoprotease hemicellulase Endoprotease Xylanase 0061 The amount of enzyme is chosen to ensure sufficient Peptidase beta-glucanase activity and avoid developing bitter notes. The amount used Peptidase beta-amylase depends on the activity of the enzyme, this information is Peptidase cellulase usually known, else it can be tested easily. The amount of Peptidase hemicellulase Peptidase Xylanase enzyme also depends on the amount of Substrate (protein or serine endopeptidase beta-glucanase carbohydrate) and there should be a ratio of 0.5:20 to 3:20 of serine endopeptidase beta-amylase enzyme: substrate (0.5 to 3 parts enzyme for 20 parts of sub serine endopeptidase cellulase strate), for example 1:20 of enzyme:substrate (CeremixTM, serine endopeptidase hemicellulase serine endopeptidase Xylanase Novozymes, Bagsvaerd, Denmark, has an activity of 300 L-glutamine-amido-hydrolase beta-glucanase Beta-Glucanase Units (BGU) per gram of enzyme; Visco L-glutamine-amido-hydrolase beta-amylase ZymeTM, Novozymes, Bagsvaerd, Denmark, has an activity of L-glutamine-amido-hydrolase cellulase 100 Fungal Beta-Glucanase Units FBG per gram of enzyme; L-glutamine-amido-hydrolase hemicellulase L-glutamine-amido-hydrolase Xylanase AlcalaseTM, Novozymes, Bagsvaerd, Denmark, has an activ Subtilisin peptidase beta-glucanase ity of 2.4 Anson untis (AU) per gram of enzyme; Umam Subtilisin peptidase beta-amylase izymeTM, Amano, Nagoya, Japan, has an activity of 70 U Subtilisin peptidase cellulase (Units by LGG method, LGG-L-Leucyl-Glycyl-Glycine); Subtilisin peptidase hemicellulase Flavorpro 373TM, a Glutaminase, Biocatalysts, Cardiff, UK, Subtilisin peptidase Xylanase has an activity of 30 Glutaminase Units (GU)). 0062 Useful amounts of enzyme units per gram starting 0054. A useful combination is a 1,3-beta-glucan-gluco material are indicated for some type of enzymes below. hydrolase (EC 3.2.1.58) with protease selected from a serine 0063 Beta-Glucanase Units (BGU) per gram starting endopeptidase, a peptidase/protease, or a Subtilisin peptidase material (liquified spinach slurry) 0.03 to 15 BGU, for (EC 3.4.21.62). example 0.1 to 3 BGU. 0055 As 1,3-beta-glucan-gluco-hydrolase, for example, 0064. Fungal Beta-Glucanase Units FBG per gram start without limitation, one or more of CeremixTM (Novozymes, ing material, 0.002 to 3 FBG, for example, 0.01 to 1 FBG. Bagsvaerd, Denmark) or ViscozymeTM (Novozymes, Bags 0065 Anson untis (AU) per gram starting material, 0.0002 Vaerd, Denmark) may be used. to 0.02 AU, for example 0.0005 to 0.01. 0056. As protease/peptidase/glutaminase, for example, 0066 U (Units by LGG method, LGG=L-Leucyl-Glycyl without limitation, one or more of AlcalaseTM, a serine Glycine) per gram starting material 0.007 to 0.7 U, for endopeptidase (Novozymes, Bagsvaerd, Denmark), Umam example, 0.01 to 0.1 U are used. US 2015/0342236 A1 Dec. 3, 2015

0067. Glutaminase Units (GU) per gram starting material, are well known in the art, for example, without limitation, 0.00075 to 0.075 GU, for example, 0.001 to 0.02 GU are used. about 70° C., about 90° C. or higher for 30 min When heating 0068. The amount of enzyme will vary depending on above about 100° C., for example, about 121° C. for 30 min, enzyme and conditions it is used in. The necessary amount heating has to be performed under pressure, usually about can be easily determined by trying out different amounts and 12-15 psi). testing the effect of the resulting product in a sensory evalu 0080. The pH during fermentation should be from about ation as described herein. pH 5 to about pH 7. If the pH is below 5, Lactobacillus 0069. The hydrolysate of the spinach slurry hydrolyzed by plantarum will grow very slowly and usually not sufficiently. one or more of proteolytic and optionally one or more of During fermentation the pH will lower to about pH4 or lower, carbohydrase enzymes may be used directly as a salt-enhanc for example about pH 5 to about pH 3.5. ing ingredient. Usually it will be heat-inactivated before use I0081. Afterwards, the pasteurized fermentation broth may by a final heat treatment (sterilization or pasteurization) high be filtered to remove any larger particles and may be concen and long enough to inactivate enzymes and optionally micro trated, for example by evaporation, including boiling at for organisms, as detailed hereinunder. example up to about 100° C. 0070 Alternatively, the hydrolysate is subjected to a fer mentation. Form of Use I0082. The salt-enhancing ingredient may be used as such Fermentation or in filtered and/or concentrated form. Alternatively, the 0071 Fermentation is performed with a Lactobacillus concentrated salt-enhancing ingredient may be used as a bacterium, for example, Lactobacillus plantarum. Other Lac paste or powder or spray-dried by methods well known in the tobacillus species may also be useful, for example, L. casei, art. For the spray-dried salt-enhancing ingredient, well L. brevis and L. helveticus may also be useful. known carriers and anti-caking agents may be added. 0072 An overnight culture of Lactobacillus may be used, I0083) Optional filtering may be performed by any suitable or the hydrolysate may be directly inoculated from a Lacto filtering method, such methods are well known in the art, for bacillus clone, and the fermentation performed for a slightly example, by passing through a felt filter bag in a filter centri longer time accordingly. fuge. The filtered culture (Supernatant containing the remain 0073. The seed culture/overnight culture for the following ing Smaller Solids, minus the biomass that includes larger fermentation may be prepared by methods well-known in the undigested proteins) can be concentrated, for example con art. It may be grown overnight, for example 12 hours, at the centrated 2x by evaporation/boiling at 100° C. The resulting appropriate temperature for the microorganism. 37° C. is a concentrate's solid content can be determined using a mois Suitable temperature for L. plantarum. Any suitable medium ture analyser and can be spray-dried, for example, onto a may be selected, for example MRS broth (Difico, United suitable carrier. Many carriers are well known in the art, for States of America). example, without limitation, a potato maltodextrin carrier 0.074 The inoculated material is fermented for several (for example, a ratio of about 1:1 solids of the 2xconcentrate hours, for example, 5 hours to 48 hours, 10 hours to 30 hours, to carrier may be Suitable). Optionally an anti-caking agent or 15 hours to 25 hours. may be added, such agents are well known. A Suitable anti 0075. The fermentation with Lactobacillus is started using caking agent is, for example, tricalciumphosphate (TPC); the hydrolysate as fermentation broth and adding a sufficient about 0.5% (wit/wt) based on total weight of the 2xconcen volume of an overnight seed culture at a pH of at least 5 or trate would be a suitable amount. higher, for example a pH of 5 to 7. Fermentation is allowed to I0084. The final form of the salt-enhancing ingredient may proceed until the pH has lowered to 25 at least pH 5.5 or be chosen according to methods well known in the art and will lower, for example pH 5.5 to pH 4.5. depend on the particular food application. For liquid foods, 0076. The fermentation temperature is chosen to accom for example soups, the salt-enhancing ingredient can be used modate the microorganism. Useful temperature ranges for without further processing in its liquid form. For dry appli Lactobacilli and in particular L. plantarum include, for cations such as crackers, the spray-dried concentrated Salt example, from about 20° C. to about 40°C., from about 30° C. enhancing ingredient can be used. to about 40° C., or from about 35 to about 40° C., with an I0085. The salt-enhancing ingredient may be directly optimum of about 36°C. to about 38°C. At a low temperature added to food products, or may be provided as part of a flavour the growth rate will be low, at a high temperature the micro composition for flavouring food products. organism will be killed or reduced in numbers. I0086 Flavour compositions contain the salt-enhancing 0077. The fermentation container should be minimally ingredient and optionally one or more food grade excipient. stirred to ensure proper mixing but at the same time ensure Suitable excipients for flavour compositions are well known that the bacteria can grow anaerobically (Lactobacili are in the art and include, for example, without limitation, Sol facultative anaerobic but usually grow faster under anaerobic vents (including water, alcohol, ethanol, oils, fats, vegetable conditions, aerotolerance may be manganese-dependent). oil, and miglyol), binders, diluents, disintegranting agents, 0078. The fermented product can be used directly as a lubricants, flavoring agents, coloring agents, preservatives, salt-enhancing ingredient, but usually will be followed by a antioxidants, emulsifiers, stabilisers, flavor-enhancers, final heat treatment (sterilization or pasteurization) high and Sweetening agents, anti-caking agents, and the like. long enough to inactivate enzymes and microorganisms. Examples of such carriers or diluents for flavours may be 0079. Usually the hydrolyzed or the fermented product found e.g. in “Perfume and Flavor Materials of Natural Ori will be heat-inactivated before use, for example by heating gin.” S. Arctander, Ed., Elizabeth, N.J., 1960; in “Perfume from about 60° C. to about 121°C. or higher for sufficiently and Flavor Chemicals'. S. Arctander, Ed., Vol. I & II, Allured long to inactivate enzymes and bacteria (for example, without Publishing Corporation, Carol Stream, USA, 1994; in “Fla limitation, any pasteurization or sterilization method, which vourings’’. E. Ziegler and H. Ziegler (ed.), Wiley-VCH Wein US 2015/0342236 A1 Dec. 3, 2015

heim, 1998, and “CTFA Cosmetic Ingredient Handbook'. J. salty Snacks (potato chips, crisps, nuts, tortilla-tostada, pret M. Nikitakis (ed.), 1st ed., The Cosmetic, Toiletry and Fra Zels, cheese Snacks, corn Snacks, potato-Snacks, ready-to-eat grance Association, Inc., Washington, 1988. popcorn, microwaveable popcorn, caramel corn, pork rinds, 0087. The flavour composition may contain additional fla nuts), crackers (Saltines, Ritz type), “sandwich-type' Vour ingredients including flavour compounds, flavours from cracker Snacks, breakfast cereals, cheeses and cheese prod natural sources including botanical sources and including ucts including cheese analogues (reduced sodium cheese, ingredients made by fermentation. The flavour composition pasteurized processed cheese (food, Snacks & spreads), may have any Suitable form, for example liquid or Solid, wet savoury spreads, cold pack cheese products, cheese sauce or dried, or in encapsulated form bound to or coated onto products), meats, aspic, cured meats (ham, bacon), luncheon/ carriers/particles or as a powder. breakfast meats (hotdogs, cold cuts, sausage), soya-based 0088. If the salt-enhancing ingredient is added as an products, tomato products, potato products, dry spice or sea unconcentrated liquid, about 0.25 ppm (0.00025% wit/wt) to soning compositions, liquid spice or seasoning compositions about 400 ppm (0.4% wt/wt) are usually enough in reduced or including pesto, marinades, and Soup-type/meal-alternative low Sodium applications, for example, without limitation, in beverages, and vegetablejuices including tomato juice, carrot Soups and topical food applications such as chips, crisps and juice, mixed vegetable juices and other vegetable juices. Snacks. 0.095 Processed foods include margarine, peanut butter, 0089. Notably, lower concentrations of the salt enhancer Soup (clear, canned, cream, instant, UHT), gravy, canned (for example, below 25 ppm, below 50 ppm, below 100 ppm, juices, canned vegetable juice, canned tomato juice, canned or below 200 ppm) were found to have a better salt-enhancing fruit juice, canned juice drinks, canned vegetables, pasta effect than higher concentrations, for example of about 300 sauces, frozen entrees, frozen dinners, frozen hand-held ppm. However, depending on the food product, a higher con entrees, dry packaged dinners (macaroni & cheese, dry din centration may be needed, for example at least 25 ppm, at ners-add meat, dry salad/side dish mixes, dry dinners-with least 50 ppm, at least 100 ppm, at least 200 ppm, or at least meat). Soups may be in different forms including condensed 300 ppm. In addition to a decreasing 20 salt-enhancing effect, wet, ready-to-serve, ramen, dry, and bouillon, processed and higher concentrations may introduce off-tastes that depend pre-prepared low-sodium foods. ing on the food product the salt-enhancing ingredient will be 0096. Depending on the food product, for food products used in may be not acceptable to the consumer. that contain about 10 to 100%, for example 25 to 50%, less 0090. For most topical food applications, about 0.5 to Sodium than a comparable food product (for example about 50 ppm are usually sufficient. “reduced sodium” products with 25% reduction, or “light in 0091. When using a concentrate (for example by distilla sodium” products with a 50% reduction), the salt-enhancing tion) or a spray-dried salt-enhancing ingredient, the concen ingredient may be employed as follows: a useful concentra trations indicated need to be adjusted with an appropriate tion for most food applications may be, for example, about factor to take into account of the concentration change in the 0.001% to about 0.015% (wit/wt) based on the unconcentrated salt-enhancing ingredient. salt-enhancing ingredient. Alternatively, for example, 25 to 300 ppm or 0.002% to 0.03% (wit/wt) based on a spray-dried Food Products 2xconcentrate may be used. 0092. The term food product is used in a broad meaning to 0097. The salt enhancer may be used in unconcentrated or include any product placed into the oral cavity but not neces concentrated form or the concentrate may be formulated into sarily ingested, including, without limitation, food, bever a paste or powderby methods known in the art. In this case the ages, nutraceuticals and dental care products including mouth amount to be used has to be adjusted accordingly. Flavour wash. compositions such as spices are often more concentrated, for 0093 Food products include cereal products, rice prod example a 10xconcentrate, and the concentration will be ucts, pasta products, ravioli, tapioca products, Sago products, adjusted higher accordingly. baker's products, biscuit products, pastry products, bread 0098. The NaCl concentration in common food products products, confectionery products, dessert products, gums, with a regular NaCl concentration varies with most products chewing gums, chocolates, ices, honey products, treacle ranging from about 0.5% to about 5% (wit/wt) NaCl. Season products, yeast products, salt and spice products, savory ing or products used as seasoning. Such as croutons, sauces or products, mustard products, vinegar products, sauces (condi salad dressings that are employed in a small amount (to be ments), processed foods, cooked fruits and vegetable prod applied to, for example, salad or noodles), have a concentra ucts, meat and meat products, meat analogues/substitutes, tion of for example from about 2% to about 5% (wit/wt) NaCl. jellies, jams, fruit sauces, egg products, milk and dairy prod Soups usually contain about 0.6% to about 1.25% (wit/wt) ucts, cheese products, butter and butter Substitute products, NaCl. Salty crackers and meat products (salami, ham, bacon) milk Substitute products, soy products, edible oils and fat usually contain about 2% to about 4% (wit/wt) NaCl. Cereals products, medicaments, beverages, juices, fruit juices, Veg usually contain about 0.6 to 3% (wit/wt) NaCl. Products that etablejuices, food extracts, plant extracts, meat extracts, con need to be reconstituted (dry soups) usually range in the diments, nutraceuticals, gelatins, tablets, lozenges, drops, concentration ranges indicated after reconstitution. emulsions, elixirs, syrups, and combinations thereof. 0099 For low sodium products containing even less NaCl 0094. Of particular interest are, without limitation, food than products with reduced sodium content (353 mg per serv products traditionally high in Sodium salt with a reduced ing), the amount of the salt-enhancing ingredient may have to Sodium salt concentration, including condiments and sauces be increased. (cold, warm, instant, preserved, sate, tomato, BBQ Sauce, 0100 For food products with added KCl, depending on the Ketchup, mayonnaise and analogues, bechamel), gravy, chut food product and said ingredients, the concentration of KCl ney, salad dressings (shelf stable, refrigerated), batter mixes, may be from about 0.1% or about 0.2% up to about 1%, up to Vinegar, pizza, pasta, instant noodles, french fries, croutons, about 1.5%, up to about 2% (wit/wt), or higher, depending on US 2015/0342236 A1 Dec. 3, 2015

how much the Sodium concentration is reduced. A KCl con -continued centration of about 0.25% to about 1.5% (wit/wt), for example about 0.5% to about 1.5% (wit/wt)KC1 will be useful for most Spinach leaves slurry samples Enzymes low Sodium products. A range to which the NaCl concentra Powder - control tion may usefully be reduced for most applications is, for Powder - total a) U&V example, about 0.25% (wit/wt) to about 2.5% (wit/wt), or from Powder - total b) UV&G about 0.125% to about 1.25% (wit/wt). The amount of the Powder - volatiles - control salt-enhancing ingredient to be added to the food product as Powder - non-volatiles - control an ingredient will depend on the concentration of KCl used, Powder - non-volatilesia) U&V and the specific food product including the particular base Powder - non-volatiles b) UV&G and flavour. A useful concentration for most food applications may be, for example, about 0.001% to about 0.015% (wit/wt) ViscozymeTM (Novozymes, Bagsvaerd, Denmark) has an based on the unconcentrated Salt-enhancing ingredient. Alter activity of 100 Fungal Beta-Glucanase Units FBG per gram natively, for example, 25 to 300 ppm or 0.002% to 0.03% of enzyme; per gram starting material, 0.1 FBG are used. (wt/wt) based on a spray-dried 2xconcentrate may be used. UmamizymeTM (Amano, Nagoya, Japan) has an activity of 70 0101 The salt-enhancing ingredient may be used in un U (Units by LGG method, LGG-L-Leucyl-Glycyl-Glycine); concentrated form or the concentrate may be formulated into per gram starting material, 0.07 U are used. a paste or powder or spray-dried salt-enhancing ingredient by (O112 A Glutaminase, Flavorpro 373TM (Biocatalysts, methods known in the art. In this case, the amount to be used Cardiff, UK), may be used as a proteolytic enzyme. Flavorpro has to be adjusted accordingly. 373TM has an activity of 30 Glutaminase Units (GU); per gram 0102 The appropriate concentration of the salt-enhancing starting material, 0.0075 GU are used. ingredient can be easily tested by an organoleptic titration. 0113. Enzymatic hydrolysis was allowed to proceed for 20 to 24 hours at 50° C. while stirring at 150 rpm to form a This technique is well known in the field of sensory analysis. hydrolysate. EXAMPLES 0114. The hydrolysate was then cooled to 37° C. and inoculated with an overnight culture of a strain of Lactoba 0103 Unless otherwise indicated, percentages or ratios cillus plantarum (cell density of about 10° cells/g) in a con are given as wit/wt. centration of 0.3% overnight culture per hydrolyzed material/ fermentation broth. Example 1 0115 The inoculated material underwent fermentation for 0104 Enzymatic hydrolysis of spinach and fermentation about 24 hours (or until the pH had lowered to about pH 4) at of the spinach hydrolysate 37°C. under minimal stifling. Fermentation was followed by 0105 Various different samples were prepared as indi a final heat treatment of 121° C. for 30 min cated in the table below. 0106 Fresh spinach leaves were finely chopped with a Example 2 food processor. Water was added to the chopped spinach in a 1:9 ratio (waterispinach) and the slurry was liquified in the Sensory Evaluation food processor. 0116. The samples of example 1 were organoleptically 0107. A part of the liquified spinach slurry was separated evaluated by trained flavorists in a 50% sodium-reduced, by distillation into a volatile and a non-volatile/pot residue fat-free chicken broth (sodium 480 mg/serving) in two fraction. sample or control concentrations (5 ppm and 300 ppm). The 0108. In parallel, instead of liquified slurry of fresh spin chicken broth was served warm (about 37°C.) for tasting and ach, liquified slurry of dehydrated (air dried) spinach powder all samples were presented blinded (so their identity cannot was used in a concentration of 15% in water, and achieved be identified by panelists). similar results. 0117. Each of the samples was compared to a control 0109 The liquified slurry of fresh or dehydrated spinach, processed as described in example but without the enzyme or their non-volatile fractions, were heated to 50° C. and the treatment, and an increased salt taste intensity over the con following samples were prepared by adding the following trol was rated as follows: + slightly salty, ++ salty, +++ strong digestive enzymes in the indicated concentrations (w/w) to salty taste. the slurry: 0118. The results are shown in the table below. 0110 a) ViscozymeTM (0.1%) and UmamizymeTM (0.1%) UV 0111 b) UmamizymeTM (0.1%), ViscozymeTM (0.1%) Enhancement Enhancement Spinach leaves slurry of saltiness of saltiness and Glutaminase Flavorpro 373TM (0.025%) UVG samples Enzymes 300 ppm 5 ppm Fresh - total - control Spinach leaves slurry Samples Enzymes Fresh - totala) U&V ------Fresh - total b) UV&G ------Fresh - total - control Fresh - volatiles - control Fresh - totala) U&V Fresh - non-volatiles - Fresh - total b) UV&G control Fresh - volatiles - control Fresh - non-volatiles a) U&V ------Fresh - non-volatiles - control Fresh - non-volatiles b) UV&G ------Fresh - non-volatiles a) U&V Powder - control Fresh - non-volatiles b) UV&G Powder - totala) U&V ------US 2015/0342236 A1 Dec. 3, 2015

-continued be limited to any single embodiment, but rather construed in breadth and scope in accordance with the recitation of the Enhancement Enhancement attached claims. Spinach leaves slurry of saltiness of saltiness 1. A process of forming a salt-enhancing ingredient com samples Enzymes 300 ppm 5 ppm prising the steps of Powder - total b) UV&G ------(i) forming an aqueous slurry of spinach plant material, and Powder - volatiles - control — (ii) forming a hydrolysate of spinach plant material by Powder - non-volatiles - Subjecting it to an enzymatic hydrolysis using one or control more proteolytic enzymes. Powder - non-volatilesia) U&V ------2. The process of claim 1, wherein the formed salt-enhanc Powder - non-volatiles b) UV&G ------ing ingredient is inactivated by heating. 3. The process according to claim 1, wherein the one or more proteolytic enzymes are selected from the group con Salt Enhancement When Compared to Controls without sisting of proteinase, peptidase, and glutaminase. Enzyme Treatment 4. The process according to claim 1, wherein the one or 0119) All panelists found that the cultured hydrolyzate of more proteolytic enzymes comprise both endopeptidase and the fresh spinach leaves sample, as well as that of dry spinach exopeptidase activity. powder, showed a good salt-enhancing effect at a concentra 5. The process according to claim 1, 4 wherein the one or tion of 5 ppm resulting in a pleasantly upfront salty taste very more proteolytic enzymes comprise an enzyme preparation similar in its temporal flavor profile to sodium chloride, with from Aspergillus oryzae (UmamizymeTM) and the hydrolysis out any off-taste. The effect was decreased somewhat in the is performed at 40° C. to 60° C. samples containing 300 ppm, and, a slight metallic off-taste 6. The process according to claim 1, wherein the hydroly (++) was noted by some panelists, especially in the powder sate is formed by Subjecting the Spinach plant material to an nonvolatiles a) sample with UV but without G. The other enzymatic hydrolysis using one or more carbohydrase samples treated with U and V but not G also had a very slight enzymes in parallel or Subsequent to enzymatic hydrolysis by off-taste (+), compare results in the table below. the one or more proteolytic enzymes. 7. The process according to claim 1, wherein the hydroly sate is Subjected to fermentation using a Lactobacillus; Off-taste Off-taste preferably a Lactobacillus microorganism selected from Spinach leaves slurry samples Enzymes 5 ppm 300 ppm the group consisting of L. plantarum, L. casei, L. brevis Fresh - totala) U&V and L. helveticus. Fresh - total b) UV&G 8. The salt-enhancing ingredient formed by the process Fresh - non-volatiles a) U&V ---- according to claim 1. Fresh - non-volatiles b) UV&G 9. The salt-enhancing ingredient according to 1 claim 9 Powder - total a) U&V -- Powder - total b) UV&G which is concentrated at least 1.5 times by removing water. Powder - non-volatilesia) U&V -- 10. The salt-enhancing ingredient according to claim 8. Powder - non-volatiles b) UV&G wherein the salt-enhancing ingredient is spray-dried. 11. A flavor composition for food products comprising the Comparison UV & UVG, presence of off-tastes salt-enhancing ingredient according to claim 8, and one or more food grade excipient. Example 3 12. The flavor composition according to claim 11 wherein the concentration of the salt-enhancing ingredient of claim 9 is 0.25 to 400 ppm based on the use of the unconcentrated Sensory Evaluation salt-enhancing ingredient. 0120. The evaluation was performed as essentially as 13. A food product comprising the salt-enhancing ingredi described in example 2 leaving out the carbohydrate enzyme ent according to claim 8, which is present at a concentration (sample without “V”). When compared to samples with V of 0.25 to 400 ppm based on the use of the unconcentrated carbohydrate enzyme, the increase in Saltiness was found to salt-enhancing ingredient. be less pronounced. 14. The food product of claim 13 which is a reduced or low 0121 While the processes, ingredients and food products sodium food product, preferably with a sodium chloride con have been described above in connection with certain illus centration is 0.15% (wit/wt) to 3% (wit/wt). trative embodiments, it is to be understood that other similar 15. The reduced or low sodium food product according to embodiments may be used or modifications and additions claims 14, additionally comprising KCl. may be made to the described embodiments for performing 16. A method of providing a food product enhanced in the same function(s). Further, all embodiments disclosed are saltiness the method comprising the step of admixing to a not necessarily in the alternative, as various embodiments food product a salt-enhancing ingredient according to claim may be combined to provide the desired characteristics. 8. Variations can be made by one having ordinary skill in the art 17. The method of claim 16 wherein the food product is a without departing from the scope of the disclosure. There reduced or low sodium food product. fore, the processes, ingredients and food products should not k k k k k