Sourdough products

Abstract

The present application provides a sourdough product comprising waste bread or derivative thereof fermented in the presence of one or more proteolytic enzymes, in particular one or more added proteolytic enzymes. Also provided herein is a method for preparing a sourdough product comprising the steps of: a) mixing waste bread or derivative thereof with water thereby obtaining a mixture; and b) fermenting the mixture obtained in a) in the presence of one or more proteolytic enzymes at a temperature from 25.0°C to 50.0°C for a period of from 8 hours to 100 hours, thereby obtaining a liquid sourdough product.

Description

[0001] SOURDOUGH PRODUCTS

[0002] FIELD OF THE INVENTION

[0003] The present invention provides methods for obtaining sourdough products for use in the preparation of bakery products, the sourdough products obtained by such methods and the use thereof.

[0004] BACKGROUND OF THE INVENTION

[0005] The food industry is currently being driven by consumers to become more conscious about food waste, food recycling and sustainable processes. Among food waste, bakery residues and in particular bread waste are generated in large quantities.

[0006] The amount of bread wasted daily worldwide, throughout its entire lifecycle, from production to distribution, is estimated to be hundred of tons, therefore representing both economic and environmental issues. Over the last decade, many researchers attempted to recycle waste bread by, for example, the production / extraction, through microbial fermentation, of bioactive compounds for pharmaceutical or food industries, of biofuels, or of enzymes, or by using them directly as feed for livestock. However, recycling surplus bread for human food purposes, such as new bakery ingredients, would be a more sustainable practice.

[0007] Attempts to recycle bread waste have been made.

[0008] It is the aim of the present invention to provide sourdoughs products that allow an improved recyclability of waste bread.

[0009] SUMMARY OF THE INVENTION

[0010] The present inventors found to their surprise that that the performances of sourdough products based on waste bread, and more particularly the leavening power of sourdough products, can be improved by adding a proteolytic enzyme during the sourdough fermentation, as compared to a control. As a result thereof, such sourdough products allow to prepare baked products with an increased specific volume and alveolation, as compared to control baked products. Accordingly, a first aspect provides a sourdough product comprising waste bread or derivative thereof, fermented in the presence of one or more proteolytic enzymes, in particular in the presence of one or more added proteolytic enzymes. In particular embodiments, said one or more proteolytic enzymes, in particular said one or more added proteolytic enzymes, are one or more bacterial and / or fungal proteolytic enzymes, preferably one or more proteolytic enzymes obtained from a Bacillus sp. and / or an Aspergillus sp., more preferably a proteolytic enzyme obtained from Bacillus amyloliquefaciens and / or a proteolytic enzyme obtained from Aspergillus oryzae.

[0011] In particular embodiments, said waste bread or derivative thereof is fermented by one or more strains of lactic acid bacteria and one or more strains of yeast.

[0012] In particular embodiments, said one or more strains of lactic acid bacteria are chosen from the list consisting of Lactiplantibacillus plantarum, Levilactobacillus brevis, Lactobacillus sanfranciscensis, Limosilactobacillus fermentum, Lactobacillus curvatus, Lacticaseibacillus casei, Furfurilactobacillus rossiae and / or Lactobacillus pentosus, preferably said one or more strains of lactic acid bacteria are Lactiplantibacillus plantarum.

[0013] In particular embodiments, said one or more strains of yeast are chosen from the list consisting of Saccharomyces cerevisiae, Kluyveromyces lactis, Kluyveromyces marxianus, Kazachstania bulderi, Kazachstania exigua and / or Kazachstania humilis, preferably said one or more strains of yeast are chosen from Saccharomyces cerevisiae.

[0014] In particular embodiments, said waste bread or derivative thereof, has not been treated with one or more proteolytic enzymes and / or amylolytic enzymes prior to fermentation.

[0015] In particular embodiments, said sourdough product is a liquid sourdough product.

[0016] In particular embodiments, said liquid sourdough product has a pH from 3.5 to 5.5, preferably from 3.8 to 5.2.

[0017] In particular embodiments, said sourdough product has a total titratable acidity (TTA) from 2.0 to 15.0, preferably from 2.0 to 10.0, more preferably from 2.0 to 5.0.

[0018] A further aspect provides a method for preparing a sourdough product comprising the steps of: a) mixing waste bread or derivative thereof with water, thereby obtaining a mixture; and b) fermenting the mixture obtained in a) in the presence of one or more proteolytic enzymes, in particular in the presence of one or more added proteolytic enzymes, at a temperature from 25.0°C to 50.0°C for a period of from 8 hours to 100 hours, thereby obtaining a liquid sourdough product.

[0019] In particular embodiments, said one or more proteolytic enzymes, in particular said one or more added proteolytic enzymes, are one or more bacterial and / or fungal proteolytic enzymes, preferably one or more proteolytic enzymes obtained from a Bacillus sp. and / or an Aspergillus sp., more preferably a proteolytic enzyme obtained from Bacillus amyloliquefaciens and / or a proteolytic enzyme obtained from Aspergillus oryzae.

[0020] In particular embodiments, said sourdough product is fermented by one or more strains of lactic acid bacteria and one or more strains of yeasts, preferably wherein said one or more strains of lactic acid bacteria are chosen from the list consisting of Lactiplantibacillus plantarum, Levilactobacillus brevis, Lactobacillus sanfranciscensis, Limosilactobacillus fermentum, Lactobacillus curvatus, Lacticaseibacillus casei, Furfurilactobacillus rossiae and / or Lactobacillus pentosus and / or wherein said one or more strains of yeast are chosen from the list consisting of Saccharomyces cerevisiae, Kluyveromyces lactis, Kluyveromyces marxianus, Kazachstania bulderi, Kazachstania exigua and / or Kazachstania humilis.

[0021] A further aspect provides the use of a sourdough product as taught herein as an ingredient in the preparation of a food product, preferably a bakery or patisserie product.

[0022] A further aspect provides a baked product comprising a sourdough product as taught herein.

[0023] DETAILED DESCRIPTION OF THE INVENTION

[0024] Before the present method and devices used in the invention are described, it is to be understood that this invention is not limited to particular methods, components, or devices described as such methods, components, and devices may, of course, vary. It is also to be understood that the terminology used herein is not intended to be limiting, since the scope of the present invention will be limited only by the appended claims.

[0025] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although any method and material similar or equivalent to those described herein may be used in practice or testing of the present invention, the preferred methods and materials are now described.

[0026] In this specification and the appended claims, the singular forms "a", "an", "the" include both the singular and the plural, unless the context clearly indicates otherwise.

[0027] The terms "comprising", "comprises" and "comprised of" as used herein are synonymous with "including", "includes" or "containing", "contains", and are inclusive or open-ended and do not exclude additional, non-recited members, elements or method steps. Where this description refers to a product or process which "comprises" specific features, parts or steps, this refers to the possibility that other features, parts or steps may also be present, but may also refer to embodiments which only contain the listed features, parts or steps. The terms "comprising", "comprises" and "comprised of" also include the term "consisting of".

[0028] The enumeration of numeric values by means of ranges of figures comprises all values and fractions in these ranges, as well as the cited end points.

[0029] The terms "about" and "approximately" as used when referring to a measurable value, such as a parameter, an amount, a time period, and the like, is intended to include variations of + / 10% or less, preferably + / -5% or less, more preferably + / -1% or less, and still more preferably + / -0.1% or less, of and from the specified value, in so far as the variations apply to the invention disclosed herein. It should be understood that the value to which the term "about" or "approximately" refers per se has also been disclosed.

[0030] The inventors of the present invention have surprisingly found that it was possible to improve the performances, and more particularly the leavening power, of sourdough products based on waste bread by adding one or more proteolytic enzymes during the sourdough fermentation. As a result thereof, such sourdough products allow to prepare baked products with an increased specific volume and alveolation, as compared to control baked products.

[0031] It is therefore a first object of the present invention to provide sourdough products, particularly food-grade sourdough products, with improved leavening power comprising waste bread or a derivative thereof, wherein said waste bread or derivative thereof is fermented in the presence of one or more, such as one, two or three, proteolytic enzymes, in particular in the presence of one or more added proteolytic enzymes. In particular embodiments, the sourdough product is suitable for human consumption.

[0032] The term "waste bread", "wasted bread" or "WB", in the context of the present invention, refers to any kind of bakery product waste. Bread waste may be for example crusts and / or external layer of bread that has been removed from loaves in sandwich bread production, returned staled bread that became unfit for sale or bread having suffered from production problems such as over-fermentation, slight over-baking or any situation leading to bread with lack of volume or unacceptable shape. In particular embodiments, the waste bread is optionally dried, and ground and / or milled to reduce its size to a mean size of less than 5.0 mm, preferably less than 2.0 mm, more preferably less than 1.0 mm. In particular embodiments, the resulting powder, particularly waste bread powder, has a maximal particle size of less than 5.0 mm, preferably less than 2.0 mm, more preferably less than 1.0 mm. In particular embodiments, the waste bread may be dried prior to grounding and / or milling. It will be obvious for the skilled person that the waste bread should be free of any contaminants that would make the sourdough unsuitable for human consumption.

[0033] In particular embodiments, the waste bread or derivative thereof is a waste bread powder (WBP). The term "waste bread powder", "wasted bread powder" or "WBP", in the context of the present invention, refers to a powder obtained after drying and grinding bread waste. Bread waste may be any kind of bakery product waste. Bread waste may be for example crusts and / or external layer of bread that has been removed from loaves in sandwich bread production, returned staled bread that became unfit for sale or bread having suffered from production problems such as over-fermentation, slight over-baking or any situation leading to bread with lack of volume or unacceptable shape. Preferably, the waste bread powder is obtained starting from the crust of sandwich bread.

[0034] The drying of the waste bread may be performed by any suitable method known in the art. Nonlimiting examples of drying methods are hot air drying (e.g. conventional or convection oven), sun drying, contact drying (e.g. drums), infrared drying, freeze-drying, spinning mill with circulating hot air or fluid-bed drying. In a preferred embodiment, the waste bread (e.g. bread crusts) is hot air dried. For example, the waste bread may be dried at a temperature between 40.0 and 80.0°C, preferably between 45.0 and 65.0°C, for a period between 1 and 10 hours, preferably between 3 and 6 hours, such as in an oven.

[0035] The dried waste bread may be ground using any suitable method or apparatus known in the art. After grinding the waste bread powder has preferably a mean size (e.g. mean particle size) of less than 5.0 mm, such as less than 4.0 mm or less than 3.0 mm, preferably less than 2.0 mm, more preferably less than 1.0 mm.

[0036] The expression "fermented in the presence of one or more proteolytic enzymes", in the context of the present invention, particularly refers to a microbial fermentation of a mixture comprising a fermentation substrate, wherein the fermenting mixture comprises added proteolytic enzyme(s), i.e. proteolytic enzymes that are not present in the fermentation substrate and which are not produced by the fermenting micro-organisms during the fermentation.

[0037] The expression "fermented in the presence of one or more added proteolytic enzymes", in the context of the present invention, particularly refers to a microbial fermentation of a mixture comprising a fermentation substrate, wherein the fermenting mixture comprises added proteolytic enzyme(s), i.e. proteolytic enzymes that are not present in the fermentation substrate and which are not produced by the fermenting micro-organisms during the fermentation.

[0038] In the context of the present invention, the fermentation substrate is a baked good or bread product, particularly a waste bread product or a derivative thereof.

[0039] The term "proteolytic enzyme", in the context of the present invention, refers to an enzyme that catalyses the hydrolysis of proteins or (poly)peptides to smaller (poly)peptides or amino acids. Alternative names include protease, peptidase or proteinase. Any proteolytic enzyme may be used to achieve the goal of the present invention. In preferred embodiments, the proteolytic enzyme is active at temperatures and pHs close to the temperatures and pHs of the fermentation of the waste bread, particularly the fermentation of the waste bread powder.

[0040] In preferred embodiments, the proteolytic enzyme, in particular the added proteolytic enzyme, is of microbial origin, such as of bacterial or fungal origin. In particular embodiments, the bacterial proteolytic enzyme is a Bacillus proteolytic enzyme. In particular embodiments, the fungal proteolytic enzyme is an Aspergillus proteolytic enzyme. Non-limiting examples of suitable proteolytic enzymes are the neutral protease of Bacillus amyloliquefaciens and protease(s) of Aspergillus oryzae.

[0041] In particular embodiments, said one or more proteolytic enzymes, in particular said one or more added proteolytic enzymes, are one or more bacterial and / or fungal proteolytic enzymes, preferably a proteolytic enzyme obtained from Bacillus amyloliquefaciens and / or a proteolytic enzyme obtained from Aspergillus oryzae. In particular embodiments, said one or more proteolytic enzymes is a neutral protease obtained from Bacillus amyloliquefaciens or a protease obtained from Aspergillus oryzae.

[0042] Preferably, said waste bread or derivative thereof has not been treated with one or more proteolytic enzymes and / or amylolytic enzymes prior to being fermented.

[0043] In particular embodiments, the waste bread or derivative thereof, more in particular the waste bread powder, is fermented in the absence of any other enzyme than said one or more proteolytic enzymes, in particular said one or more added proteolytic enzymes. In other embodiments, the waste bread or derivative thereof, more in particular the waste bread powder, is fermented in the presence of one or more proteolytic enzymes and in the presence of one or more additional enzymes, such as an (alpha)-amylase or a glucoamylase. In particular embodiments, the waste bread or derivative thereof, more in particular the waste bread powder, is fermented by one or more strains of lactic acid bacteria and one or more strains of yeast in the presence of one or more proteolytic enzymes, in particular in the presence of one or more added proteolytic enzymes.

[0044] In the context of the present invention, the term "lactic acid bacteria" refers to any food-grade bacteria producing lactic acid as major metabolite of the carbohydrate fermentation. Nonlimiting examples of suitable strains of lactic acid bacteria are strains of the genus Lactiplantibacillus, Lactobacillus, Leuconostoc, Pediococcus, Lactococcus, Streptococcus, Bifidobacterium, Enterococcus, Oenococcus and / or Weissella. Non limiting examples of suitable species of lactic acid bacteria are strains of Lactiplantibacillus plantarum, Levilactobacillus brevis, Lactobacillus sanfranciscensis, Limosilactobacillus fermentum, Lactobacillus curvatus, Lacticaseibacillus casei, Furfurilactobacillus rossiae and / or Lactobacillus pentosus. In particular embodiments, the lactic acid bacteria strain(s) is / are one or more strains of Lactiplantibacillus plantarum.

[0045] In the context of the present invention, the term "yeast" refers to any food-grade yeast capable of producing carbon dioxide, particularly during fermentation of cereal based material, such as a dough, or, in the context of the present invention, during fermentation of waste bread, more in particular, waste bread powder. Non-limiting examples of suitable yeast strains are strains of the genus Saccharomyces, Kluyveromyces, Kazachstania, Candida, Torulopsis, Yarrowia and / or Pichia. Non-limiting examples of suitable yeast strains are strains of Saccharomyces cerevisiae, Kluyveromyces lactis, Kluyveromyces marxianus, Kazachstania bulderi, Kazachstania exigua and / or Kazachstania humilis. In particular embodiments, the yeast strain(s) is / are Saccharomyces cerevisiae.

[0046] In particular embodiments, the cell density of the lactic acid bacteria in the sourdough product according to the present invention ranges from 5.0*108to 1.0*1010, preferably from 0.7*109to 5*109, more preferably from 0.8*109to 2*109. In particular embodiments, the cell density of the yeast in the sourdough product according to the present invention ranges from 1.0*107to 5.0*108, preferably from 5.0*107to 1.0*108.

[0047] The sourdough product may be in any form. In particular embodiments of the present invention the sourdough product is a liquid sourdough product. In particular embodiments, such as if the sourdough product is a liquid sourdough product, the sourdough product has a pH from 3.5 to 5.5, preferably from 3.8 to 5.2. In particular embodiments, the sourdough product has a total titratable acidity (TTA) from 2.0 to 15.0, preferably from 2.0 to 10.0, such as from 2.0 to 8.0, from 2.0 to 7.0, from 2.0 to 6.0, more preferably from 2.0 to 5.0, such as from 2.5 to 5.0. The Total Titratable Acidity of a sourdough product is a product property commonly known to the person skilled in the art. The TTA refers to the amount (expressed in ml) of 0.1 N NaOH needed to bring the pH of 10 g of product to 8.3.

[0048] In a further aspect, the present application relates to methods for preparing a sourdough product as described herein. In particular embodiments, the method for preparing a sourdough product comprises the steps of: a) mixing waste bread or a derivative thereof, such as waste bread powder, with a suitable liquid, preferably water, thereby obtaining a mixture; and b) fermenting the mixture obtained in a) in the presence of one or more proteolytic enzymes, in particular one or more added proteolytic enzymes, at a temperature from 25.0°C to 40.0°C, preferably from 25.0°C to 35.0°C, for a period from 8 hours to 100 hours, preferably from 16 to 72 hours thereby obtaining a sourdough product, preferably a liquid sourdough product.

[0049] It is understood that the waste bread or derivative thereof is the fermentation substrate for the microbial fermentation of step b).

[0050] The waste bread or derivative thereof may be mixed with other ingredients than water prior to fermentation. Accordingly, in particular embodiments, the water is part of an aqueous composition.

[0051] In particular embodiments, said waste bread or derivative thereof has not been treated with one or more proteolytic enzymes and / or amylolytic enzymes prior to fermentation (i.e. prior to step b)). In particular, said waste bread or derivative thereof has not been treated with one or more added proteolytic enzymes and / or amylolytic enzymes prior to fermentation (i.e. prior to step b)).

[0052] In particular embodiments of the method as taught herein, the sourdough product is fermented by one or more strains of lactic acid bacteria and / or one or more strains of yeast, with the lactic acid bacteria and the yeast strains as described elsewhere herein. The person skilled in the art will understand that these are living bacteria and / or yeast. It is thus understood that step a) further comprises (i) adding the one or more strains of lactic acid bacteria and / or the one or more strains of yeast to the mixture, typically thereby initiating the fermentation of the mixture of step b), as well as (ii) adding one or more proteolytic enzymes to the mixture, with the one or more proteolytic enzymes as described elsewhere herein.

[0053] In particular embodiments, the one or more strains of lactic acid bacteria are one or more strains of the genus Lactiplantibacillus, Lactobacillus, Leuconostoc, Pediococcus, Lactococcus, Streptococcus, Bifidobacterium, Enterococcus, Oenococcus and / or Weissella.

[0054] In particular embodiments, the one or more strains of yeast are one or more strains of the genus Saccharomyces, Kluyveromyces, Kazachstania, Candida, Torulopsis, Yarrowia and / or Pichia.

[0055] In particular embodiments, the one or more strains of lactic acid bacteria are one or more strains of Lactiplantibacillus plantarum, Levilactobacillus brevis, Lactobacillus sanfranciscensis, Limosilactobacillus fermentum, Lactobacillus curvatus, Lacticaseibacillus casei, Furfurilactobacillus rossiae and / or Lactobacillus pentosus. Preferably, the one or more strains of lactic acid bacteria are one or more strains of Lactiplantibacillus plantarum.

[0056] In particular embodiments, the one or more strains of yeast are one or more strains of Saccharomyces cerevisiae, Kluyveromyces lactis, Kluyveromyces marxianus, Kazachstania bulderi, Kazachstania exigua and / or Kazachstania humilis. Preferably, the one or more strains of yeast are one or more strains of Saccharomyces cerevisiae.

[0057] In the method as taught herein, the waste bread or derivative thereof, particularly the waste bread powder, may firstly be suspended in a suitable liquid, preferably water, thereby obtaining a (liquid) mixture / slurry. The dry matter of the (liquid) mixture comprising waste bread and liquid, such as water, is preferably from 5.0 to 35.0% (w / w), more preferably from 7.0% to 30.0% (w / w), more preferably from 10.0% to 25.0% (w / w).

[0058] Said (liquid) mixture may be fermented by the addition of bacteria and / or yeast to said mixture. Said mixture, particularly liquid mixture, further comprises one or more proteolytic enzymes, as described elsewhere herein. In certain embodiments, the one or more proteolytic enzymes are added to the mixture, particularly liquid mixture, in an amount ranging from 0.05 to 500 units per gram waste bread or derivative thereof, such as from 0.1 to 100 units per kg waste bread or derivative thereof, wherein one unit corresponds to the amount of proteolytic enzyme which catalyzes the liberation from hemoglobin or casein of fragments, soluble in trichloroacetic acid, equivalent to 1 pmol of tyrosine per minute, particularly at pH 6.0-7.0 and 37°C. In particular embodiments, said mixture is fermented by one or more strains of lactic acid bacteria in an amount from 106to 108bacterial cells / g of (liquid) mixture, preferably from 0.5 * 107to 1.5 * 107bacterial cells / g of (liquid) mixture and / or one or more strains of yeast in an amount from 104to 10syeast cells / g of (liquid) mixture, preferably from 0.5 * 105to 1.5 * 105yeast cells / g of (liquid) mixture. The fermentation / incubation may be performed for a period from 8 to 100 hours, preferably from 16 to 72h and / or at a temperature from 25.0°C to 50.0°C, from 25.0°C to 40.0°C, preferably from 25.0°C to 35.0°C.

[0059] In a preferred embodiment, the living cells (i.e. lactic acid bacteria and / or yeast) are added under the form of cells having been first cultivated in another culture medium to obtain a starter culture. More preferably, the different strains of lactic acid bacteria and / or yeast are separately cultivated in another culture medium to obtain a starter culture. in particular embodiments of the method as taught herein, the fermentation is performed with agitation to ensure uniform dispersion of the substrate (i.e. the mixture comprising waste bread or derivative thereof and water) and the one or more strains of lactic acid bacteria and one or more strains of yeast within the fermentation mixture, in particular embodiments, the fermentation is performed without addition / insufflation of air or oxygen to the fermentation mixture. in particular embodiments, the method as taught herein provides in a liquid sourdough product, being the end product of the fermentation step. In particular embodiments, the liquid sourdough product is characterized by having a pH from 3.5 to 5.5, preferably from 3.8 to 5.2. Suitable compounds known to the skilled person may be used to adjust the pH to the desired value.

[0060] In particular embodiments, the method as taught herein provides in a sourdough product, the end product of the fermentation step, characterized by a dry matter at the end of the fermentation of from 5.0 to 35.0% (w / w), preferably from 7.0 to 30.0% (w / w), more preferably from 10.0 to 25.0% (w / w).

[0061] A further aspect provides a sourdough product obtained or obtainable by the method as taught herein.

[0062] A further aspect provides a method for improving the characteristics of a food product, preferably a baked product, comprising the steps of: preparing or providing a sourdough product as taught herein; adding said sourdough product to a dough or batter, and; baking said dough or batter comprising the sourdough product as taught herein thereby obtaining a baked product.

[0063] Advantageously the sourdough product as taught herein improves physical properties of the baked product such as the volume (e.g. specific volume) and the alveolation of the crumb. In particular embodiments, the volume (e.g. specific volume) of the baked product obtained by or obtainable by the method as taught herein or using the sourdough product as taught herein is improved by at least 5.0%, at least 10.0%, such as by at least 15.0%, or at least 20.0%, compared to a control baked product prepared with a sourdough product based on waste bread or derivative thereof, which has been fermented in the absence of an added proteolytic enzyme.

[0064] A further aspect provides the use of a sourdough product as taught herein as an ingredient in the preparation of a food product, preferably a baked product such as a bakery or patisserie product.

[0065] A further aspect provides a baked product, such as a fresh baked product, comprising a sourdough product as taught herein. In particular embodiments, the baked product is prepared from a dough or batter comprising the sourdough composition as taught herein. In particular embodiments, the baked product is obtained by or obtainable from the method as taught herein.

[0066] In particular embodiments, the baked product is a bakery or patisserie, such as, but not limited to, those selected from the group consisting of bread, soft rolls, bagels, donuts, Danish pastry, hamburger rolls, pizza, pita bread, ciabatta, sponge cakes, cream cakes, pound cakes, muffins, cupcakes, steamed cakes, waffles, brownies, cake donuts, yeast raised donuts, baguettes, rolls, crackers, cookies, pie crusts, rusks and / or other baked products. Preferably, the baked product is bread, baguettes and / or rolls, more preferably bread.

[0067] The person skilled in the art will understand that the particular embodiments of the products as taught herein are also applicable to the methods and uses as taught herein and vice versa.

[0068] While the invention has been described in conjunction with specific embodiments thereof, it is evident that many alternatives, modifications, and variations will be apparent to those skilled in the art in light of the foregoing description. Accordingly, it is intended to embrace all such alternatives, modifications, and variations as follows in the spirit and broad scope of the appended claims.

[0069] The above aspects and embodiments are further supported by the following non-limiting examples.

[0070] EXAMPLES

[0071] Example 1: Materials

[0072] Strains

[0073] Lactiplantibacillus plantarum SD69.B2 and Saccharomyces cerevisiae SD69.B3 were isolated from a live sourdough collected in Liguria (Italy).

[0074] Media

[0075] L. plantarum was routinely propagated in modified MRS (mMRS) broth at 30°C for 24h. The composition of the MRS broth is: peptone (10 g / L), "Lab-Lemco" Powder (8 g / L), Yeast extract (4 g / L), Glucose (20 g / L), Sorbitan mono-oleate (1 ml), Di-potassium hydrogen phosphate (2 g / L), Sodium acetate BHjO (5 g / L), Tri-ammonium citrate (2 g / L), Magnesium sulphate 7HzO (0,2 g / L), Manganese sulphate 4HzO (0,05 g / L).

[0076] S. cerevisiae was routinely propagated in Sabouraud broth at 30°C for 24h. The composition of the Sabouraud broth is: Casein peptone (5 g / L), Meat peptone (5 g / L), D(+) - Glucose (20 g / L).

[0077] Starter cultures

[0078] When used as starters for fermentation, cells were grown till the late exponential phase of growth and thereafter harvested by centrifugation at 10,000 rpm for 10 min at 4°C, washed twice and resuspended in sterile physiological solution (NaCI 0.9%, w / v). Starters cultures were diluted to a lactic acid bacteria and yeast cell density of 109CFU / mL and 107CFU / mL of starter culture, respectively).

[0079] Waste bread powder (WBP)

[0080] Waste bread powder was prepared using bread crusts obtained from the cutting of the outer layer of loaves baked for the production of soft sandwich bread. Crusts were dried in an oven at 55°C for 6 hours. Dried breads crumb were ground in a mixer (Microton MB 550, Kinematica) into crumbs with a mean size below 1.0 mm and frozen until use.

[0081] Enzymes

[0082] - GAMYL : glucoamylase obtained from Trichoderma reesei

[0083] - FAMYL : alpha-amylase obtained from Aspergillus oryzae

[0084] - BPROT : bacterial neutral protease obtained from Bacillus amyloliquefaciens

[0085] - FPROT : fungal protease obtained from Aspergillus oryzae

[0086] Example 2: sourdoughs

[0087] Sourdough were prepared using the composition of table 1.

[0088] Table 1: sourdoughs compositions

[0089] Process

[0090] Ingredients of table 1 were mixed in a blender (Kenwood Mutipro, UK) for 5 min. Sourdoughs SI to S5 were incubated directly at 30°C for 24 h under stirring conditions (130 rpm). Sourdough S6 was obtained in two steps: first the mixture with the enzyme but without lactic acid bacteria and yeasts was allowed to rest at 30°C for 12 hours. Thereafter L. plantarum and S. cerevisiae were added and the mixture was incubated at 30°C for 24 h under stirring conditions (130 rpm).

[0091] Sourdoughs evaluation The following parameters of the sourdoughs were evaluated:

[0092] - Cell density: 10 ml of sample were mixed with 90 ml of sterile physiological solution (NaCI 0.9%, w / v) and homogenized with a Stomacher 400 lab blender (Seward Medical) followed by serial dilution in physiological solution. LAB and yeasts were enumerated on agar plates and incubation at 30°C for 48 h using mMRS agar medium, supplemented with cycloheximide (0.1 g / L) (Sigma-Aldrich, USA) for lactic acid bacteria and Sabouraud agar medium, supplemented with chloramphenicol (0.1 g / L) (Sigma-Aldrich, USA) for yeasts. 2 dilutions were counted and the average was calculated.

[0093] - pH: the pH of the sourdough was measured after 24h of fermentation

[0094] - Total Titratable Acidity (TTA): The TTA refers to the amount (expressed in ml) of 0.1 N NaOH needed to bring the pH of 10 g of product to 8.3.

[0095] - Leavening power: 150 g of Type 00 wheat flour, 75 ml of water and 25 ml of sourdough were mixed for 7 minutes at high speed. The dough was placed in a 500 ml Bormioli jar and the increase in volume was measured with a ruler after 6 hours at 30°C. The value for the leavening powers is expressed relative to the control SI without enzymes that was set to 100

[0096] Results

[0097] Table 2

[0098] The results show that, by treating the WBP with a proteolytic enzyme during the fermentation of the sourdough it was possible to obtain sourdoughs with a higher leavening power compared to the control, i.e a sourdough composition prepared in the absence of a proteolytic enzyme.

[0099] Example 3: Breads Breads were prepared using the ingredients of Table 3.

[0100] Table 3

[0101] Process

[0102] Ingredients were mixed using a continuous high-speed mixer (60 x g, dough mixing time 7 min) to obtain doughs having a dough yield of 160. Doughs were fermented for 6 hours at 30°C and subsequently baked at 230°C for 35 min (Omega 2, Bongard, Italy). Doughs were prepared in triplicates and each bread was analysed twice.

[0103] Bread analysis

[0104] The specific volume of three replicate breads was measured using a BVM 6600 Perten Instrument. The test mode settings were as follows: test duration 60 s, cylinder calibration with no attachments. Specific volume analysis was carried out using VolCalc software, which measured volume (mL), height (mm), width (mm), depth (mm), and weight (g) of the loaves.

[0105] The alveolation of the bread (% of area covered by alveoli) was measured using Image J software (Java, 1.52t version, 2020).

[0106] Both volumes and alveolation are expressed relative to the control Bl that was set to 100.

[0107] The results are given in Table 4.

[0108] Table 4

[0109] The use of a sourdough products as described herein shows a positive effect on specific volume and alveolation. More particularly, breads prepared using sourdough products as described herein have a higher specific volume and alveolation compared to breads prepared using control sourdough products, i.e a sourdough composition prepared in the absence of a proteolytic enzyme.

Claims

CLAIMS1. A sourdough product comprising waste bread or derivative thereof fermented in the presence of one or more added proteolytic enzymes.

2. The sourdough product according to claim 1, wherein said one or more added proteolytic enzymes are one or more bacterial and / or fungal proteolytic enzymes, preferably one or more proteolytic enzymes obtained from a Bacillus sp. and / or an Aspergillus sp., more preferably a proteolytic enzyme from Bacillus amyloliquefaciens and / or a proteolytic enzyme obtained from Aspergillus oryzae.

3. The sourdough product according to claim 1 or 2, wherein said waste bread or derivative thereof is fermented by one or more strains of lactic acid bacteria and one or more strains of yeast.

4. The sourdough product according to claim 3, wherein said one or more strains of lactic acid bacteria are chosen from the list consisting of Lactiplantibacillus plantarum, Levilactobacillus brevis, Lactobacillus sanfranciscensis, Limosilactobacillus fermentum, Lactobacillus curvatus, Lacticaseibacillus casei, Furfurilactobacillus rossiae and / or Lactobacillus pentosus, preferably said one or more strains of lactic acid bacteria are Lactiplantibacillus plantarum.

5. The sourdough product according to claim 3 or 4, wherein said one or more strains of yeast are chosen from the list consisting of Saccharomyces cerevisiae, Kluyveromyces lactis, Kluyveromyces marxianus, Kazachstania bulderi, Kazachstania exigua and / or Kazachstania humilis, preferably said one or more strains of yeast are chosen from Saccharomyces cerevisiae.

6. The sourdough product according to any one of claims 1 to 5, wherein said waste bread or derivative thereof has not been treated with one or more proteolytic enzymes and / or amylolytic enzymes prior to fermentation.

7. The sourdough product according to any one of claims 1 to 6, wherein said sourdough product is a liquid sourdough product.

8. The sourdough product according to claim 7 , wherein said liquid sourdough product has a pH from 3.5 to 5.5, preferably from 3.8 to 5.2.

9. The sourdough product according to any one of claims 1 to 8, wherein said sourdough product has a total titratable acidity (TTA) from 2.0 to 15.0, preferably from 2.0 to 10.0, more preferably from 2.0 to 5.0.

10. A method for preparing a sourdough product comprising the steps of: a) mixing waste bread or derivative thereof with water thereby obtaining a mixture; andb) fermenting the mixture obtained in a) in the presence of one or more added proteolytic enzymes at a temperature from 25.0°C to 50.0°C for a period of from 8 hours to 100 hours, thereby obtaining a liquid sourdough product.

11. The method according to claim 10, wherein said one or more added proteolytic enzymes are one or more bacterial and / or fungal proteolytic enzymes, preferably one or more proteolytic enzymes obtained from a Bacillus sp. and / or an Aspergillus sp., more preferably a proteolytic enzyme from Bacillus amyloliquefaciens and / or a proteolytic enzyme obtained from Aspergillus oryzae.

12. The method according to claim 10 or 11, wherein said sourdough product is fermented by one or more strains of lactic acid bacteria and one or more strains of yeast, preferably wherein said one or more strains of lactic acid bacteria are chosen from the list consisting of Lactiplantibacillus plantarum, Levilactobacillus brevis, Lactobacillus sanfranciscensis, Limosilactobacillus fermentum, Lactobacillus curvatus, Lacticaseibacillus casei, Furfurilactobacillus rossiae and / or Lactobacillus pentosus and / or wherein said one or more strains of yeast are chosen from the list consisting of Saccharomyces cerevisiae, Kluyveromyces lactis, Kluyveromyces marxianus, Kazachstania bulderi, Kazachstania exigua and / or Kazachstania humilis.

13. Use of a sourdough product according to any of claims 1 to 9 as an ingredient in the preparation of a food product, preferably a bakery or patisserie product.

14. A baked product comprising a sourdough product according to any of claims 1 to 9.

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