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  • A23J3/00—Working-up of proteins for foodstuffs
  • A23J3/30—Working-up of proteins for foodstuffs by hydrolysis
  • A23J3/32—Working-up of proteins for foodstuffs by hydrolysis using chemical agents
  • A23J3/34—Working-up of proteins for foodstuffs by hydrolysis using chemical agents using enzymes
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  • A23L27/24—Synthetic spices, flavouring agents or condiments prepared by fermentation
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  • C12Y304/00—Hydrolases acting on peptide bonds, i.e. peptidases (3.4)
  • C12Y304/25—Threonine endopeptidases (3.4.25)
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  • C12Y305/00—Hydrolases acting on carbon-nitrogen bonds, other than peptide bonds (3.5)
  • C12Y305/01—Hydrolases acting on carbon-nitrogen bonds, other than peptide bonds (3.5) in linear amides (3.5.1)
  • C12Y305/01002—Glutaminase (3.5.1.2)
• • A—HUMAN NECESSITIES
  • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
  • A23V—INDEXING SCHEME RELATING TO FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES AND LACTIC OR PROPIONIC ACID BACTERIA USED IN FOODSTUFFS OR FOOD PREPARATION
  • A23V2002/00—Food compositions, function of food ingredients or processes for food or foodstuffs

Definitions

• a high amount of sodium intake is considered to be detrimental to health and therefore there is a desire to reduce the amount of sodium chloride (NaCl) in food products, without reducing the desired salty taste at the same time.
• the salty taste is very important to the perceived flavour intensity and profile, especially for savory food products.
• KCl Potassium Chloride
• a process of forming a salt-enhancing ingredient comprising the steps of (i) forming an aqueous slurry of spinach plant material, and (ii) forming a hydrolysate of spinach plant material by subjecting it to an enzymatic hydrolysis using one or more proteolytic enzymes. (2) The process as described under item (1), wherein the formed salt-enhancing ingredient is inactivated by heating.
• Lactobacillus microorganism is selected from the group consisting of L. plantarum, L. casei, L. brevis and L. helveticus.
• a flavor composition for food products comprising the salt-enhancing ingredient as described under any one of items (9) to (1 1) and one or more food-grade excipient.
• concentration of the salt-enhancing ingredient of claim 9 is 0.25 to 400 ppm based on the use of the unconcentrated salt-enhancing ingredient.
• Useful concentrations for the salt-enhancing ingredient (unconcentrated) in flavor compositions or food products include 0.25 to 300 ppm, 0.25 to 200 ppm, 0.5 to 100 ppm, and 0.5 to 20 ppm.
• a food product comprising the salt-enhancing ingredient as described under any one of items (9) to (1 1).
• salt-enhancing is meant the effect of an ingredient on the salty taste in food which is found more pronounced (stronger, enhanced) in its taste intensity and/or longer in its duration as analyzed by trained panellists sensitive to salty taste, when comparing food comprising an ingredient with a salt-enhancing effect to food without an added salt- enhancing ingredient.
• the enhanced intensity of the perception of salty taste by the salt-enhancing ingredient can be increased by additionally using a carbohydrase enzyme, either in parallel or consecutively, in its formation.
• a higher concentration may be, for example, about 50 ppm or more, about 100 ppm or more, about 200 ppm or more, or about 300 ppm or more
• glutaminase it is advantageous to include glutaminase in the enzymatic treatment.
• the enhanced intensity of the perception of salty taste can be further increased by an optional fermentation step employing Lactobacillus bacteria, for example, Lactobacillus plantarum. Spinach
• “Spinach” as used herein refers to the green leaves and/or stalks of a flowering plant in the family of Amaranthaceae (formerly known as Chenopodiaceae), including Spinacia oleracea, and the closely related species Beta vulgaris (also known as chard, spinach beet, silverbeet or perpetual spinach); Atriplex spec, including A. hortensis (also known as orache, French spinach, or mountain spinach); and Chenopodium spec, including C. bonus- henricus and C. album (also known as Wild spinach, Fat hen, Good King Henry or Lincolnshire spinach).
• Spinach includes three to four types, Savoy, Flat/Smooth leaf spinach, Semi-savoy, and Baby.
• Savoy has dark green, crinkly and curly leaves.
• the Bloomsdale and the Tyee are popular savoy type varieties.
• Flat/smooth leaf spinach has broad smooth leaves that are easier to clean and is grown for canned, frozen or processed foods.
• Semi-savoy is a hybrid variety with slightly crinkled leaves. Semi-savoy includes Five Star, a widely grown variety. Baby is of the flat-leaf type and its leaves are usually no longer than three inches.
• Examples of spinach varieties include, without limitation, America, Bloomsdale or Bloomsdale Long Standing, Dominant, Giant Winter, Horenso, Medania, Sigmaleaf, Space, Trinidad, Tyee (savoy type), Olympia, Melody, Winter Bloomsdale, Bordeaux, Koto, Lazio, Vienna, Marathon, Seven R, Baker, Cascade, Olympia, Polka, Rainier, Shasta, Wo her, Bossanova, Bolero, Coho, Brown, Rainier, Rhythm 9, Hybrid #7, Skookum, Bejo 1369, Splendor, Indian Summer, Avon, Correnta, Nordic IV, Savoy Supreme, Space, Spokane, Springfield, Steadfast, Unipak 12, Mazurka, Chinook II, Hybrid 424, St. Helens, Baker, Imperial Express and Imperial Star.
• Exemplary useful enzyme classes include proteolytic enzymes that hydrolyze bonds in a protein, and optionally, a carbohydrase.
• Proteolytic enzyme preparations usually contain proteinases, which hydrolyze proteins to form small peptides, and peptidases, which hydrolyze small proteins or peptides, usually to release amino acids from their terminal ends.
• proteinases and peptidases with both endopeptidase and exopeptidase activity are included in such preparations, to efficiently break down a protein both from within and from the ends of each protein and resulting peptide.
• proteolytic enzymes include, without limitation, an enzyme with one or more of the following activities: protease, peptidase, glutaminase (including, without limitation, L- glutamine-amido-hydrolase (EC 3.5.1.2)), endoprotease, serine endopeptidase, subtilisin peptidase (EC 3.4.21.62).
• proteolytic enzymes are useful as well, and a great variety is known and available; some additional types and examples are given below.
• Proteolytic enzymes are currently classified in six groups including serine protease, threonine protease, cysteine protease, aspartic acid protease, metalloprotease, and glutamic acid protease.
• Proteolytic enzymes can cut at the end of a protein (exopeptidases) or attack internal peptide bonds of a protein (endopeptidases).
• Exopeptidases include, without limitation, aminopeptidases, carboxypeptidases, and carboxypeptidase A.
• Endopeptidases include, without limitation, trypsin, chymotrypsin, pepsin, papain, and elastase.
• Proteolytic enzymes (EC 3.4 and EC 3.5) are classified by an EC number (enzyme commission number), each class comprises various known enzymes of a certain reaction type.
• EC 3.4 comprises enzymes acting on peptide bonds (peptidases/proteinases) and EC 3.5 comprises enzymes that act on carbon-nitrogen bonds other than peptide bonds.
• Examples for EC 3.4 include, without limitation, the following: aminopeptidase (EC 3.4.1 1), dipeptidase (3.4.13), dipeptidyl-peptidase (3.4.14), peptidyl-dipeptidase (3.4.15), serine-carboxypeptidase (3.4.16), metallocarboxypeptidase (3.4.17), cysteine- carboxypeptidase (3.4.18), omegapeptidase (3.4.19), serine-endopeptidase (3.4.21), cysteine-endopeptidase (3.4.22), aspartate-endopeptidase (3.4.23), metalloendopeptidase (3.4.24), threonine-endopeptidase (3.4.25).
• aminopeptidase EC 3.4.1 1
• dipeptidase 3.4.13
• dipeptidyl-peptidase 3.4.14
• peptidyl-dipeptidase 3.4.15
• Examples for EC 3.5 include, without limitation, proteolytic enzymes that cleave in linear amides (3.5.1), for example, without limitation, glutaminase (EC 3.5.1.2).
• proteolytic enzymes are commercially available; the following proteolytic enzymes are available from Sigma-Aldrich: Achromopeptidase, Aminopeptidase, Ancrod, Angiotensin Converting Enzyme, Bromelain, Calpain, Calpain I , Calpain II, Carboxypeptidase A, Carboxypeptidase B, Carboxypeptidase G, Carboxypeptidase P , Carboxypeptidase W, Carboxypeptidase Y, Caspase, Caspase 1, Caspase 2, Caspase 3, Caspase 4, Caspase 5, Caspase 6, Caspase 7,Caspase 8, Caspase 9, Caspase 10, Caspase 13, Cathepsin B, Cathepsin C, Cathepsin D, Cathepsin G, Cathepsin H, Cathepsin L,
• proteolytic enzymes described herein can be combined with a carbohydrase to increase the salt impact of the formed salt-enhancing ingredient.
• Useful enzyme combinations include, without limitation, combinations wherein at least one proteolytic enzyme is combined with at least one carbohydrase.
• carbohydrase enzymes to break down carbohydrate plant material include, without limitation, carbohydrases with one or more of the following activities: beta-glucanase (including, without limitation, 1,3-beta-glucan-gluco-hydrolase (EC 3.2.1.58)), beta-amylase, cellulase, hemicellulase, xylanase.
• beta-glucanase including, without limitation, 1,3-beta-glucan-gluco-hydrolase (EC 3.2.1.58)
• beta-amylase beta-amylase
• cellulase hemicellulase
• hemicellulase hemicellulase
• xylanase xylanase
• a useful combination is a l ⁇ -beta-glucan-gluco-hydrolase (EC 3.2.1.58) with protease selected from a serine endopeptidase, a peptidase/protease, or a subtilisin peptidase (EC 3.4.21.62).
• 1,3-beta-glucan-gluco-hydrolase for example, without limitation, one or more of CCeerreemmiixxTMTM ((NNoovvoozzyymmeess, Bagsvaerd, Denmark) or ViscozymeTM (Novozymes, Bagsvaerd, Denmark) may be used.
• protease/peptidase/glutaminase for example, without limitation, one or more of
• AlcalaseTM a serine endopeptidase (Novozymes, Bagsvaerd, Denmark), UmamizymeTM, a protease/peptidase (Amano, Nagoya, Japan), or Flavorpro 373TM, a subtilisin peptidase (Biocatalysts, Cambridge, UK), may be used.
• Enzymatic hydrolysis is performed under conditions suitable for all enzymes employed.
• the temperature and pH should be within a suitable range for hydrolysis to occur to the desired degree.
• the incubation length will vary accordingly, with shorter incubations when conditions are nearer to the optimum conditions. Usually 1 to 48 hours will be sufficient, for example, 10 to 24 hours.
• Necessary ions, if required or beneficial for the chosen enzyme(s), should be present, as the skilled person will be aware.
• Stirring the incubation mix for example 50 to 500 rpm, or 100 to 200 rpm, usually improves the hydrolysis.
• Some enzymes tolerate stirring better than others. Tolerance towards one factor often depends on the other factors. Such information on suitable conditions is readily available for many enzymes and otherwise can be easily determined.
• a number of enzyme preparations including CeramixTM, AlcalaseTM, ViscozymeTM , and UmamizymeTM, will work well in a liquified slurry of spinach in water at a temperature from 40°C to 55°C, for example about 45°C to about 55°C, without pH adjustment or any added co-factors. Others may need or will benefit from pH or temperature adjustment, or additives.
• UmamizymeTM will tolerate temperatures from about 40 0 C to about 60°C, with an optimum at around 55 0 C.
• UmamizymeTM originates from Aspergillus oryzae and is rich in endopeptidase and exopeptidase activity.
• the amount of enzyme is chosen to ensure sufficient activity and avoid developing bitter notes.
• the amount used depends on the activity of the enzyme, this information is usually known, else it can be tested easily.
• the amount of enzyme also depends on the amount of substrate (protein or carbohydrate) and there should be a ratio of 0.5:20 to 3:20 of enzyme: substrate (0.5 to 3 parts enzyme for 20 parts of substrate), for example 1 :20 of enzyme: substrate (CeremixTM, Novozymes, Bagsvaerd, Denmark, has an activity of 300 Beta-Glucanase Units (BGU) per gram of enzyme; ViscozymeTM, Novozymes, Bagsvaerd, Denmark, has an activity of 100 Fungal Beta-Glucanase Units FBG per gram of enzyme; AlcalaseTM, Novozymes, Bagsvaerd, Denmark, has an activity of 2.4 Anson untis (AU) per gram of enzyme; UmamizymeTM, Amano, Nagoya, Japan, has an activity of 70 U (Units
• Beta-Glucanase Units per gram starting material (liquified spinach slurry) 0.03 to 15
• BGU for example 0.1 to 3 BGU.
• Anson untis (AU) per gram starting material 0.0002 to 0.02 AU, for example 0.0005 to
• LGG L-Leucyl-Glycyl-Glycine
• GU Glutaminase Units
• the amount of enzyme will vary depending on enzyme and conditions it is used in. The necessary amount can be easily determined by trying out different amounts and testing the effect of the resulting product in a sensory evaluation as described herein.
• the hydrolysate of the spinach slurry hydrolyzed by one or more of proteolytic and optionally one or more of carbohydrase enzymes may be used directly as a salt-enhancing ingredient. Usually it will be heat-inactivated before use by a final heat treatment (sterilization or pasteurization) high and long enough to inactivate enzymes and optionally microorganisms, as detailed hereinunder. Alternatively, the hydrolysate is subjected to a fermentation.
• Lactobacillus bacterium for example, Lactobacillus plant arum.
• Other Lactobacillus species may also be useful, for example, L. casei, L, brevis and L. helveticus may also be useful.
• Lactobacillus An overnight culture of Lactobacillus may be used, or the hydrolysate may be directly 10 inoculated from a Lactobacillus clone, and the fermentation performed for a slightly longer time accordingly.
• the seed culture/overnight culture for the following fermentation may be prepared by methods well-known in the art. It may be grown overnight, for example 12 hours, at the 15 appropriate temperature for the microorganism. 37°C is a suitable temperature for L. plantarum. Any suitable medium may be selected, for example MRS broth (Difco, United States of America).
• the inoculated material is fermented for several hours, for example, 5 hours to 48 hours, 10 20 hours to 30 hours, or 15 hours to 25 hours.
• the fermentation with Lactobacillus is started using the hydrolysate as fermentation broth and adding a sufficient volume of an overnight seed culture at a pH of at least 5 or higher, for example a pH of 5 to 7. Fermentation is allowed to proceed until the pH has lowered to 25 at least pH 5.5 or lower, for example pH 5.5 to pH 4.5.
• the fermentation temperature is chosen to accommodate the microorganism.
• Useful temperature ranges for Lactobacilli and in particular L. plantarum include, for example, from about 20°C to about 4O 0 C, from about 30°C to about 4O 0 C, or from about 35 to about 30 40 0 C, with an optimum of about 36°C to about 38°C.
• the fermentation container should be minimally stirred to ensure proper mixing but at the same time ensure that the bacteria can grow anaerobically (Lactobacilli are facultative anaerobic but usually grow faster under anaerobic conditions, aerotolerance may be manganese-dependent).
• the fermented product can be used directly as a salt-enhancing ingredient, but usually will be followed by a final heat treatment (sterilization or pasteurization) high and long enough to inactivate enzymes and microorganisms.
• the hydrolyzed or the fermented product will be heat- inactivated before use, for example by heating from about 6O 0 C to about 121 0 C or higher for sufficiently long to inactivate enzymes and bacteria (for example, without limitation, any pasteurization or sterilization method, which are well known in the art, for example, without limitation, about 70°C, about 90 0 C or higher for 30 min.
• heating has to be performed under pressure, usually about 12-15 psi).
• the pH during fermentation should be from about pH 5 to about pH 7. If the pH is below 5, Lactobacillus plantarum will grow very slowly and usually not sufficiently. During fermentation the pH will lower to about pH 4 or lower, for example about pH 5 to about pH
• the pasteurized fermentation broth may be filtered to remove any larger particles and may be concentrated, for example by evaporation, including boiling at for example up to about 100 0 C.
• the salt-enhancing ingredient may be used as such or in filtered and/or concentrated form.
• the concentrated salt-enhancing ingredient may be used as a paste or powder or spray-dried by methods well known in the art.
• well known carriers and anti-caking agents may be added.
• Optional filtering may be performed by any suitable filtering method, such methods are well known in the art, for example, by passing through a felt filter bag in a filter centrifuge.
• the filtered culture (supernatant containing the remaining smaller solids, minus the biomass that includes larger undigested proteins) can be concentrated, for example concentrated 2x by evaporation/boiling at 100°C.
• the resulting concentrate's solid content can be determined using a moisture analyser and can be spray-dried, for example, onto a suitable carrier.
• a suitable carrier for example, without limitation, a potato maltodextrin carrier (for example, a ratio of about 1 : 1 solids of the 2xconcentrate to carrier may be suitable).
• an anti-caking agent may be added, such agents are well known.
• a suitable anti-caking agent is, for example, tricalciumphosphate (TPC); about 0.5% (wt/wt) based on total weight of the 2xconcentrate would be a suitable amount.
• the final form of the salt-enhancing ingredient may be chosen according to methods well known in the art and will depend on the particular food application.
• the salt-enhancing ingredient can be used without further processing in its liquid form.
• the spray-dried concentrated salt- enhancing ingredient can be used.
• the salt-enhancing ingredient may be directly added to food products, or may be provided as part of a flavour composition for flavouring food products.
• Flavour compositions contain the salt-enhancing ingredient and optionally one or more food grade excipient.
• Suitable excipients for flavour compositions are well known in the art and include, for example, without limitation, solvents (including water, alcohol, ethanol, oils, fats, vegetable oil, and miglyol), binders, diluents, disintegranting agents, lubricants, flavoring agents, coloring agents, preservatives, antioxidants, emulsif ⁇ ers, stabilisers, flavor-enhancers, sweetening agents, anti-caking agents, and the like.
• solvents including water, alcohol, ethanol, oils, fats, vegetable oil, and miglyol
• binders include, for example, without limitation, solvents (including water, alcohol, ethanol, oils, fats, vegetable oil, and miglyol), binders, diluents, disintegranting agents, lubricants, flavoring agents, coloring agents, preservatives, antioxidants, emulsif ⁇ ers,
• the flavour composition may contain additional flavour ingredients including flavour compounds, flavours from natural sources including botanical sources and including ingredients made by fermentation.
• the flavour composition may have any suitable form, for example liquid or solid, wet or dried, or in encapsulated form bound to or coated onto carriers/particles or as a powder.
• the salt-enhancing ingredient is added as an unconcentrated liquid, about 0.25 ppm (0.00025 % wt/wt) to about 400 ppm (0.4% wt/wt) are usually enough in reduced or low sodium applications, for example, without limitation, in soups and topical food applications such as chips, crisps and snacks.
• lower concentrations of the salt enhancer (for example, below 25 ppm, below 50 ppm, below 100 ppm, or below 200 ppm) were found to have a better salt-enhancing effect than higher concentrations, for example of about 300 ppm.
• a higher concentration may be needed, for example at least 25 ppm, at least 50 ppm, at least 100 ppm, at least 200 ppm, or at least 300 ppm.
• higher concentrations may introduce off-tastes that depending on the food product the salt-enhancing ingredient will be used in may be not acceptable to the consumer.
• the concentrations indicated need to be adjusted with an appropriate factor to take into account of the concentration change in the salt-enhancing ingredient.
• food product is used in a broad meaning to include any product placed into the oral cavity but not necessarily ingested, including, without limitation, food, beverages, nutraceuticals and dental care products including mouth wash.
• Food products include cereal products, rice products, pasta products, ravioli, tapioca products, sago products, baker's products, biscuit products, pastry products, bread products, confectionery products, dessert products, gums, chewing gums, chocolates, ices, honey products, treacle products, yeast products, salt and spice products, savory products, mustard products, vinegar products, sauces (condiments), processed foods, cooked fruits and vegetable products, meat and meat products, meat analogues/substitutes, jellies, jams, fruit sauces, egg products, milk and dairy products, cheese products, butter and butter substitute products, milk substitute products, soy products, edible oils and fat products, medicaments, beverages, juices, fruit juices, vegetable juices, food extracts, plant extracts, meat extracts, condiments, nutraceuticals, gelatins, tablets, lozenges, drops, emulsions, elixirs, syrups, and combinations thereof.
• condiments and sauces cold, warm, instant, preserved, sate, tomato, BBQ Sauce, Ketchup, mayonnaise and analogues, bechamel
• gravy chutney
• salad dressings shelf stable, refrigerated
• batter mixes vinegar, pizza, pasta, instant noodles, french fries, croutons
• salty snacks potato chips, crisps, nuts, tortilla-tostada, pretzels, cheese snacks, corn snacks, potato-snacks, ready-to- eat popcorn, microwaveable popcorn, caramel corn, pork rinds, nuts
• crackers Saltines, 'Ritz' type
• "sandwich-type" cracker snacks breakfast cereals, cheeses and cheese products including cheese analogues (reduced sodium cheese, pasteurized processed cheese (food, snacks & spreads), savoury spreads, cold pack cheese products, cheese sauce products), meats, aspic, cured meats
• Processed foods include margarine, peanut butter, soup (clear, canned, cream, instant, UHT), gravy, canned juices, canned vegetable juice, canned tomato juice, canned fruit juice, canned juice drinks, canned vegetables, pasta sauces, frozen entrees, frozen dinners, frozen hand-held entrees, dry packaged dinners (macaroni & cheese, dry dinners-add meat, dry salad/side dish mixes, dry dinners-with meat). Soups may be in different forms including condensed wet, ready-to-serve, ramen, dry, and bouillon, processed and pre- prepared low-sodium foods.
• the salt-enhancing ingredient may be employed as follows: a useftil concentration for most food applications may be, for example, about 0.001% to about 0.015% (wt/wt) based on the unconcentrated salt-enhancing ingredient. Alternatively, for example, 25 to 300 ppm or 0.002% to 0.03% (wt/wt) based on a spray-dried 2x concentrate may be used.
• the salt enhancer may be used in unconcentrated or concentrated form or the concentrate may be formulated into a paste or powder by methods known in the art. In this case the amount to be used has to be adjusted accordingly.
• Flavour compositions such as spices are often more concentrated, for example a 10x concentrate, and the concentration will be adjusted higher accordingly.
• the NaCl concentration in common food products with a regular NaCl concentration varies with most products ranging from about 0.5% to about 5% (wt/wt) NaCl.
• Seasoning or products used as seasoning such as croutons, sauces or salad dressings that are employed in a small amount (to be applied to, for example, salad or noodles), have a concentration of for example from about 2% to about 5% (wt/wt) NaCl.
• Soups usually contain about 0.6% to about 1.25% (wt/wt) NaCl.
• Salty crackers and meat products (salami, ham, bacon) usually contain about 2% to about 4% (wt/wt) NaCl.
• Cereals usually contain about 0.6 to 3% (wt/wt) NaCl.
• Products that need to be reconstituted usually range in the concentration ranges indicated after reconstitution.
• the amount of the salt-enhancing ingredient may have to be increased.
• the concentration of KCl may be from about 0.1 % or about 0.2 % up to about 1%, up to about 1.5%, up to about 2% (wt/wt), or higher, depending on how much the sodium concentration is reduced.
• a KCl concentration of about 0.25 % to about 1.5% (wt/wt), for example about 0.5% to about 1.5% (wt/wt) KCl will be useful for most low sodium products.
• a range to which the NaCl concentration may usefully be reduced for most applications is, for example, about 0.25% (wt/wt) to about 2.5% (wt/wt), or from about 0.125% to about 1.25% (wt/wt).
• the amount of the salt-enhancing ingredient to be added to the food product as an ingredient will depend on the concentration of KCl used, and the specific food product including the particular base and flavour.
• a useful concentration for most food applications may be, for example, about 0.001% to about 0.015% (wt/wt) based on the unconcentrated salt-enhancing ingredient.
• 25 to 300 ppm or 0.002% to 0.03% (wt/wt) based on a spray-dried 2x concentrate may be used.
• the salt-enhancing ingredient may be used in un-concentrated form or the concentrate may be formulated into a paste or powder or spray-dried salt-enhancing ingredient by methods known in the art. In this case, the amount to be used has to be adjusted accordingly.
• the appropriate concentration of the salt-enhancing ingredient can be easily tested by an organoleptic titration. This technique is well known in the field of sensory analysis.
• Example 1 Enzymatic hydrolysis of spinach and fermentation of the spinach hvdrolvsate Various different samples were prepared as indicated in the table below.
• Fresh spinach leaves were finely chopped with a food processor. Water was added to the chopped spinach in a 1 :9 ratio (water: spinach) and the slurry was liquified in the food processor.
• a part of the liquified spinach slurry was separated by distillation into a volatile and a nonvolatile/pot residue fraction.
• the liquified slurry of fresh or dehydrated spinach, or their non-volatile fractions, were heated to 50 0 C and the following samples were prepared by adding the following digestive enzymes in the indicated concentrations (w/w) to the slurry:
• ViscozymeTM (Novozymes, Bagsvaerd, Denmark) has an activity of 100 Fungal Beta- Glucanase Units FBG per gram of enzyme; per gram starting material, 0.1 FBG are used.
• Flavorpro 373TM (Biocatalysts, Cambridge, UK), may be used as a proteolytic enzyme. Flavorpro 373TM has an activity of 30 Glutaminase Units (GU); per gram starting material, 0.0075 GU are used.
• Enzymatic hydrolysis was allowed to proceed for 20 to 24 hours at 50°C while stirring at 150 rpm to form a hydrolysate.
• the hydrolysate was then cooled to 37°C and inoculated with an overnight culture of a strain of Lactobacillus plantarum (cell density of about 10 6 cells/g) in a concentration of 0.3% overnight culture per hydrolyzed material/fermentation broth.
• the inoculated material underwent fermentation for about 24 hours (or until the pH had lowered to about pH 4) at 37°C under minimal stirring. Fermentation was followed by a final heat treatment of 121 0 C for 30 min.
• example 1 The samples of example 1 were organoleptically evaluated by trained flavorists in a 50% sodium-reduced, fat-free chicken broth (sodium 480 mg/serving) in two sample or control concentrations (5 ppm and 300 ppm). The chicken broth was served warm (about 37°C) for tasting and all samples were presented blinded (so their identity cannot be identified by panelists).

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• 2009
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