JP7507688B2 - Baking compositions containing Lactobacillus crustrum strains - Google Patents

Description

BCCM LMGP-29154

The present invention relates to the technical field of food processing. More particularly, the present invention relates to a baking composition comprising a Lactobacillus crustorum strain for producing bakery products.

Bread (including Asian steamed bread) is a traditional staple food with unique ethnic characteristics. Today, there are two main types of bread fermentation. The first type is the fermentation of wheat flour with commercial yeast. This is a simple method of producing bread, which does not take much time, but the flavor is generally poor or bland. The more traditional style is bread obtained using a pre-dough (such as sourdough or sponge) as a leavening agent. Sourdough is constantly renewed periodically, as it is restarted using a portion of the previously fermented dough, and uses a specific recipe and ripening conditions. The mother dough is then mixed with the remaining flour, water and other dough ingredients to the appropriate consistency, and then bulk fermented for a short period before final fermentation and baking. The bread or steamed bread obtained using this method is richer in flavor and more nutritious. However, this method is time consuming, and the quality of the sourdough can be inconsistent from time to time.

There are alternative ways to provide sourdough with desired characteristics and consistent quality. Two types of products may be considered:
- "Ready to use active sourdough" is an improved type of sourdough that uses adapted strains or liquid sourdough starters to initiate fermentation. These sourdoughs can be paste-like or liquid, and are generally stable and easy to process, for example in automated bread-making processes. There is enough live lactic acid bacteria and/or yeasts present to successfully ferment the dough or to start the multi-step process of sourdough.
- "Ready to use inactive powder or liquid sourdough" are used by traditional or industrial bakeries for their convenience, because of their consistent quality and ease of use. They directly avoid the long fermentation process and provide the sour taste and flavor of traditional sourdough.

Furthermore, one or several specific microbial strains may be produced, conditioned (e.g., as a dry starter) and provided to bakers to produce their own controlled sourdough.

Lactic acid bacteria play a major role in the process of sourdough fermentation. They can quickly acidify the dough through the production of organic acids such as acetic acid. They also produce other components such as ethanol, flavors, bacteriocins, exopolysaccharides and enzymes. The advantages of using sourdough fermentation are, among others: (1) improved bread flavor; (2) improved nutritional value of bread; (3) improved volume and texture of bread; (4) longer shelf life of bread. At the same time, the sourdough technique improves the processing of the dough itself, for example by reducing the sticky feeling and improving its viscoelastic properties. Lactic acid bacteria isolated from sourdough are mainly of the genera Lactobacillus, Leuconostoc, Pediococcus and/or Weissela, but the majority belong to the group of Lactobacillus. Lactic acid fermentation produces lactic acid and/or acetic acid, which impart a sour taste to sourdough. Saccharomyces cerevisiae is the most frequent yeast species in traditional sourdough. Other species of Saccharomyces, Candida , Pichia , Kluyveromyces , and/or Hansenula have also occasionally been isolated from sourdough.

Traditional sourdough production is time consuming and expensive. The quality of the sourdough is also inconsistent. Modern industry uses dominant strains to inoculate a starter dough, which can be used to produce sourdough by conditional controlled fermentation and subsequent downstream processing (e.g. drying or pasteurization). Powder, paste or liquid sourdough can be produced by these methods.

Most sourdoughs produced today are produced according to European patterns, which do not correspond well to Eastern flavor preferences. Therefore, there is a need for new sourdoughs that can improve the quality and flavor of bakery and pastry products such as bread, especially (Asian-style) steamed bread.

The present invention relates to a Lactobacillus clastrum strain deposited in the BCCM/LMG culture collection under accession number LMG P-29154, or a mutant thereof.

In a further aspect, the present invention relates to a baking composition for use in the manufacture of a bakery product comprising a Lactobacillus clastrum strain as defined herein.

In a particular embodiment, the present invention relates to a baking composition as defined herein, the baking composition being selected from a bakery product, a sourdough or a starter culture for a sourdough product.

In a particular embodiment, the present invention relates to a baking composition as defined herein further comprising one or more lactic acid bacteria strains and/or one or more yeast strains, in particular the one or more lactic acid bacteria strains are selected from acid-forming bacteria such as Leuconostoc or Lactobacillus and/or the one or more yeast strains are selected from Saccharomyces, Candida, Pichia, Kluyveromyces or Hansenula strains. More particularly, the one or more lactic acid bacteria strains are selected from Lactobacillus plantarum, Lactobacillus brevis , Lactobacillus sanfrancisciensis and/or Lactobacillus reuterii , and/or the one or more yeast strains are selected from Saccharomyces cerevisiae and Saccharomyces exiguus .

In a particular embodiment, the present invention relates to a baking composition as defined herein, preferably a sourdough or sourdough product fermented with at least one Lactobacillus clastrum strain as defined herein.

In a particular embodiment, the present invention relates to a bakery composition as defined herein, preferably comprising cereal grains or cereal pieces fermented by at least one Lactobacillus clastrum strain as defined herein, and optionally further comprising sugars and/or minerals.

In a particular embodiment, the present invention relates to a bakery composition as defined herein, which is an active or inactive sourdough or sourdough product.

In a particular embodiment, the present invention relates to a bakery composition as defined herein that is a starter culture.

In a further aspect the present invention relates to a method for producing a sourdough or sourdough product as defined herein, comprising the steps of:
- fermenting a (liquid) mixture comprising cereals and/or cereal pieces and a liquid, preferably water, with at least one Lactobacillus clastrum strain LMG p-29154 to obtain a (liquid) sourdough; and - optionally drying the (liquid) sourdough to obtain a dry composition.

In a further aspect, the present invention relates to the use of a baking composition as defined herein in the manufacture of a food product.

In a further aspect, the present invention relates to a bakery product comprising the baked composition defined herein. In particular, the bakery product is a steamed bun.

1A and 1B show morphological differences between Lactobacillus clastrum LMG P-29154 strain (FIG. 1A) and the reference L. clastrum LMG23699 strain (FIG. 1B) on a Petri dish. FIG. 1 shows the results of aroma comparison by SPME-GC-MS.

Detailed Description of the Invention

In describing the products, compositions, uses and methods of the present invention, it should be understood that the invention is not limited to the particular products, compositions, uses and methods or combinations described, since such products, compositions, uses and methods or combinations may, of course, vary. It should also be understood that the terms used herein are not intended to be limiting, since the scope of the present invention is limited only by the appended claims.

As used herein, the singular forms "a," "an," and "the" are intended to include both singular and plural referents unless the context clearly indicates otherwise.

The terms "comprising," "comprises," and "comprised of," as used herein, are synonymous with "including," "includes," or "containing," and are inclusive or open-ended and do not exclude additional, unrecited members, ingredients, or method steps. The terms "comprising," "comprises," and "comprised of," as used herein, are understood to include the terms "consisting of," "consists," and "consists of."

The recitation of numerical ranges by endpoints includes all numbers and fractions subsumed within each range, as well as the recited endpoints.

As used herein, the terms "about" or "approximately," when referring to a measurable value such as a parameter, amount, duration, and the like, are meant to encompass a variation of no more than ±10%, preferably no more than ±5%, more preferably no more than ±1%, and even more preferably no more than ±0.1% of or from the specified value, but only where such variations are appropriate for practice in the disclosed invention. It is to be understood that the value to which the "about" or "approximately" modifier refers is itself specifically and preferably disclosed.

While the term "one or more" or "at least one" is itself clear, e.g., one or more or at least one element of a group of elements, the term is intended, by way of further illustration, to specifically include reference to any one of the elements, or any two or more of the elements, e.g., any three or more, four or more, five or more, six or more, seven or more, etc. of the elements, and up to and including reference to all of the elements.

Unless otherwise defined, all terms used in disclosing the present invention, including technical and scientific terms, shall have the meaning commonly understood by one of ordinary skill in the art to which the present invention belongs. Definitions of terms are included with further guidance to better understand the teachings of the present invention.

In the following passages, different aspects of the invention are defined in more detail. Each aspect so defined may be combined with any other aspect(s), unless expressly indicated to the contrary. In particular, any feature indicated as being preferred or advantageous may be combined with any other feature(s) indicated as being preferred or advantageous.

Throughout this specification, reference to "one embodiment" or "an embodiment" means that a particular feature, structure, or characteristic described in connection with that embodiment is included in at least one embodiment of the invention. Thus, the phrases "in one embodiment" or "in an embodiment" appear in various places throughout this specification, but not necessarily all refer to the same embodiment. Furthermore, particular features, structures, or characteristics can be combined in any suitable manner in one or more embodiments, as would be apparent to one of ordinary skill in the art from this disclosure. Furthermore, while some embodiments described herein include some features but not other features included in other embodiments, combinations of features of different embodiments are intended to form different embodiments within the scope of the invention and as would be understood by one of ordinary skill in the art. For example, in the appended claims, any of the claimed embodiments can be used in any combination.

The present invention provides a particular strain of lactic acid bacteria, which is a strain of Lactobacillus clastrum, which can be advantageously used to produce starters, sourdoughs and baked products with improved properties. Preferably, the Lactobacillus clastrum strain is Lactobacillus clastrum LMG P-29154, deposited on October 2, 2015 at BCCM/LMG (Laboratorium voor Microbiologie-Bacterienverzameling; Universiteit Gent; K.L.Ledeganckstraat 35; B-9000, Ghent, Belgium) under accession number LMG P-29154 (see Table A), or a mutant thereof.

The present invention therefore relates to a Lactobacillus clastrum strain deposited in the BCCM/LMG culture collection under accession number LMG P-29154, or a mutant thereof.

As referred to herein, the term "variant" refers to microbial variants (e.g., mutants, insertion variants, and deletion variants of Lactobacillus clastrum LMG P-29154) and microbial variants having at least 90%, more preferably at least 95%, and for example, at least 96%, 97%, 98%, 99% or 99.9% total genome sequence identity.

In a further aspect, the present invention relates to a baking composition for use in the manufacture of a bakery product, comprising the Lactobacillus clastrum strain deposited in the BCCM/LMG culture collection under accession number LMG P-29154, or a variant thereof.

According to a particular embodiment, the baking composition defined herein is selected from a bakery product, a sourdough or a starter culture for a sourdough product.

According to a particular embodiment, the bakery composition defined herein further comprises one or more lactic acid bacteria strains and/or one or more yeast strains.

More specifically, the one or more lactic acid bacteria strains are selected from acid-forming bacteria such as Leuconostoc or Lactobacillus, and/or the one or more yeast strains are selected from Saccharomyces, Candida, Pichia, Kluyveromyces or Hansenula strains. More specifically, the one or more lactic acid bacteria strains are selected from Lactobacillus plantarum, Lactobacillus brevis, Lactobacillus sanfranciscensis and/or Lactobacillus reuteri, and/or the one or more yeast strains are selected from Saccharomyces cerevisiae and Saccharomyces exiguus.

The specific strains defined herein provide a unique aroma profile to bakery products produced using the baking composition according to the invention, which provides the bakery products with a unique characteristic.

According to a particular embodiment, the baked composition as defined herein is a sourdough or sourdough product. A further object of the present invention is to provide a sourdough or sourdough product produced with Lactobacillus crustrum LMG P-29154. In the context of the present invention, the term "sourdough" or "sourdough product" refers to a dough that has been fermented by lactic acid bacteria and optionally yeast. Sourdough typically has a characteristic acidic flavor due to the production of lactic acid and/or acetic acid and some minor components by the lactic acid bacteria, and a typical flavor top note, if any, produced by the yeast. Sourdough or sourdough products can be further stabilized in one or another way (e.g. through drying, pasteurization, cooling, and/or freezing) and can therefore be added to regular dough or batter, thereby replacing pre-fermentation produced in-bakery.

In a particular embodiment, the sourdough or sourdough product as defined herein comprises cereals or cereal pieces, and optionally sugar and/or minerals. Preferably, the sourdough or sourdough product comprises cereals or cereal pieces, finally mixed with sugar and/or minerals and fermented with at least one strain of Lactobacillus crustrum LMG P-29154. More particularly, such sourdough provides very good flavour and very good physical properties to baked products, such as bakery and patisserie products.

As used in this application, the term "cereal" refers in the context of the present invention to the edible parts of plants of the Poaceae family, including, but not limited to, species such as wheat, barley, oats, spelt, rye, sorghum, maize, triticale, millet, teff and/or rice. Preferably, the cereal is selected from the group of wheat, maize (corn), rice or rye. More preferred cereals are rye, rice and wheat. Even more preferred cereal is wheat.

The term "cereal pieces", in the context of the present invention, refers to all or a portion of the pieces resulting from mechanically reducing the size of grains, for example, but not limited to, through cutting, rolling, grinding, shearing, or milling, with or without fractionation (for example, but not limited to, sieving, screening, scrubbing, blowing, suction, centrifugal sieving, air sieving, electrostatic separation, or electric field separation). Preferred cereal pieces are flour, whole flours, bran, and/or any combination thereof.

The term "sugars" as used herein refers to, but is not limited to, sucrose, glucose, and compounds/products containing sucrose or glucose, such as molasses, sugar beet, sucrose, and hydrolyzed starch. As used herein, the amount of sugars refers to the sum of glucose and sucrose as measured by methods known in the art, such as high performance liquid chromatography (HPLC).

The term "minerals" refers to minerals used in the cultivation of lactic acid bacteria and includes K ⁺ , Na ⁺ , ^Mg2+ , Mn2 ⁺ , ^{Fe2 +} , _PO43- , _SO42- and ^Cl- ions. More preferably, it refers to K ⁺ and ^{Mn2 +} .

According to a particular embodiment, the present invention provides a sourdough or sourdough product, which is fermented by an additional microorganism. In this embodiment, the Lactobacillus clastrum strain used to obtain the sourdough or sourdough product taught herein, preferably Lactobacillus clastrum LMG P-29154, is combined with a conventional microorganism, i.e. a microorganism used to produce normal (conventional) sourdough. Such additional microorganism may be preferentially selected from lactic acid bacteria and/or yeasts. The lactic acid bacteria may be selected from lactic acid-forming bacteria such as Leuconostoc and Lactobacillus, preferably selected from the group consisting of Lactobacillus plantarum, Lactobacillus brevis, Lactobacillus sanfranciscensis and Lactobacillus reuteri. The yeast is advantageously selected from Saccharomyces, Candida, Pichia, Kluyveromyces or Hansenula, preferably Saccharomyces cerevisiae or Saccharomyces exiguus. In a preferred embodiment, the amount of yeast contained in the compositions taught herein, particularly the sourdough or sourdough product, is that amount required for fermentation of a dough, such as a bread dough.

In a particular embodiment, the Lactobacillus clastrum strain, preferably Lactobacillus clastrum LMG P-29154, is the only lactic acid bacteria strain present in the sourdough.

In a particular embodiment, the sourdough or sourdough product defined herein is fermented by at least a Lactobacillus clastrum strain, preferably Lactobacillus clastrum LMG P-29154.

In certain embodiments, the sourdough or sourdough product as defined herein is in liquid, paste or dry/powder form.

Sourdough products can take many forms. Liquid sourdough products are probably the most commonly used type. However, other types of sourdough products are also available, including dry or dehydrated sourdough products. Sourdough products in dry form are typically obtained by drying a liquid sourdough product. Preferably, the dry matter of the solid/powder composition is greater than 85% (w/w), preferably greater than 90%, more preferably 92% or greater. This range therefore also provides for dry matter of 86, 87, 88, 89, 91, 93, 94, 95, 96, 97, 98, 99, 100%.

According to a particular embodiment, the present invention provides a liquid sourdough product, characterized in that the liquid sourdough product has a dry matter content of 10% (w/w) to 45%, preferably 15% to 40%, more preferably 20% to 35%. This range therefore provides a dry matter content of 16, 17, 18, 19, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 41, 42, 43 or 44%.

In certain embodiments, the sourdough or sourdough product as defined herein is an active or inactive sourdough or sourdough product.

Preferably, the sourdough is an active sourdough. The term "active sourdough" means that the sourdough contains active and/or live microorganisms. The active sourdough can be used as a starter for producing a pasty or other liquid composition with the same specifications and properties as above. As with conventional active sourdoughs, the active sourdough of the invention guarantees uniformity in quality and processing. Advantageously, the range of live cells in the active sourdough is 10 ⁷ -10 ¹¹ , preferably 10 ⁷ -10 ¹⁰ , more preferably 10 ⁸ -10 ⁹ lactic acid bacteria cells/ml; and 10 ⁵ -10 ⁸ , preferably 10 ⁶ -10 ⁷ yeast cells/ml. In a particular embodiment, the active sourdough is an active liquid sourdough.

In other preferred embodiments, the sourdough is inactive. The term "inactive sourdough" means that the composition no longer contains active and/or viable microorganisms. To inactivate the sourdough as taught herein, the sourdough may be inactivated, for example, by (continuous) pasteurization at a temperature of about 55 to about 90° C. for about 10 seconds to about 60 minutes. In certain embodiments, the inactive sourdough is an inactive liquid sourdough.

In a particular embodiment, the pH of the sourdough is between 4.0 and 5.0, preferably between 4.0 and 4.5.

In certain embodiments, the sourdough of the present invention has a total titratable acidity (TTA) of higher than 12, preferably from 12 to 300, more preferably from 12 to 100, even more preferably from 12 to 50, for example from 12 to 18. Total titratable acidity is a product property commonly known to those skilled in the art. TTA refers to the amount of 0.1N NaOH (expressed in ml) required to bring 10 g of product to a pH of 8.4. The inventors have found that such a TTA results in a sourdough with improved properties, such as improved flavor.

In a particular embodiment, the present invention relates to a paste sourdough having a dry matter content of 30-70%, preferably 40-60%.

In a particular embodiment, the present invention relates to a dried sourdough product obtained by drying a liquid or paste sourdough product as taught herein. Drying of the liquid or paste sourdough can be carried out using typical drying techniques available to the skilled person. Preferably, the dried sourdough is obtained by fluidization, spray drying or drum drying of a liquid or paste composition. The preferred dry matter of the solid/powder composition is more than 90%, preferably 92%.

According to a particular embodiment, the baking composition defined herein is a starter culture for bakery products. In a particular embodiment, the baking composition defined herein is a starter culture for bakery products comprising the Lactobacillus clastrum LMG P-29154 strain. In the context of the present invention, starter culture refers to live cells of Lactobacillus clastrum LMG P-29154 and optionally other microorganisms, such as yeasts, used to produce sourdough or sourdough products. The starter culture may further comprise other strains of lactic acid bacteria and/or other strains of yeast. Preferably, the lactic acid bacteria may be selected from lactic acid-forming bacteria such as Leuconostoc or Lactobacillus, preferably selected from the group of Lactobacillus plantarum, Lactobacillus brevis, Lactobacillus sanfranciscensis and Lactobacillus reuteri. The yeast strain is advantageously selected from Saccharomyces, Candida, Pichia, Kluyveromyces or Hansenula, preferably Saccharomyces cerevisiae or Saccharomyces exiguus. The starter culture may be in dry or liquid form before use. A dry starter may be rehydrated before use, for example by suspending the starter in water containing sugar and salt. In a particular embodiment, a strain of Lactobacillus clastrum, preferably Lactobacillus clastrum LMG P-29154, is the only lactic acid bacteria strain present in the starter.

In a further aspect, the present invention relates to a method for producing a sourdough or sourdough product as defined herein, comprising the steps of:
- fermenting a (liquid) mixture comprising cereals and/or cereal pieces and a liquid, preferably water, with at least one Lactobacillus clastrum LMG p-29154 strain, thereby obtaining a (liquid) sourdough; and - optionally drying the (liquid) sourdough to obtain a dry composition.

In a particular embodiment, the mixture may further comprise additional microorganisms. Such additional microorganisms may be preferentially selected from lactic acid bacteria and/or yeasts. The lactic acid bacteria may be selected from lactic acid-forming bacteria such as Leuconostoc and Lactobacillus, preferably from the group consisting of Lactobacillus plantarum, Lactobacillus brevis, Lactobacillus sanfrancisensis and Lactobacillus reuteri. The yeasts are advantageously selected from Saccharomyces, Candida, Pichia, Kluyveromyces or Hansenula, preferably Saccharomyces cerevisiae or Saccharomyces exiguus.

In a particular embodiment, the Lactobacillus clastrum strain, preferably Lactobacillus clastrum LMG P-29154, is the only lactic acid bacteria strain present in the mixture.

In a particular embodiment, the method of the present invention provides a sourdough having a total titratable acidity (TTA) higher than 12, preferably between 12 and 300, more preferably between 12 and 100, even more preferably between 12 and 50, for example between 12 and 18. In a particular embodiment, such a TTA is obtained after 24 hours of fermentation.

The process of the present invention allows the production of industrially very useful compositions that improve the taste of baked products (obtained using sourdough) and at the same time provide baked products with satisfactory physical properties, such as a satisfactory volume.

In a preferred embodiment, the flour and water mixture is in a ratio of about 1 to 2.5 and is fermented at about 30-40°C, preferably 35-38°C, for about 12-48 hours, preferably about 12-24 hours.

In a further aspect, the present invention relates to the use of a baking composition according to the present invention in the manufacture of a food product, preferably a baked product, more preferably a bakery product or a patisserie product.

More specifically, the present invention provides for the use of the baking composition according to the present invention as part of an improver, a premix or a complete mix in the manufacture of bakery products.

As used herein, "improver" refers to compositions of the present invention that further include ingredients and/or technological aids used during manufacture and/or after baking of the baked product for their beneficial properties, including, but not limited to, appearance, volume, freshness, shelf life, color, structure or short bite of the baked product.

As used herein, the term "premix" refers to an improver composition that typically has a lower concentration of "active" ingredients than in bakery improvers. Premixes are typically used in higher dosages than improvers (wt/wt flour).

The term "complete mix" as used herein typically refers to a composition that includes all the ingredients necessary to produce a dough that can be baked to obtain a baked product, generally excluding water. In particular, when the leavening agent is a biological agent, more specifically baker's yeast, it can also be excluded from the complete mixture. A complete mix according to the present invention includes a sourdough composition according to the present invention and all the ingredients necessary to produce a dough that can be baked to obtain a baked product.

More particularly, the present invention provides the use of a sourdough product according to the present invention for stabilizing a liquid baker's yeast suspension.

In a further aspect, the present invention relates to a bakery product comprising the bakery composition according to the present invention.

A further object of the present invention is to provide bakery products obtained with sourdough containing or made with one or more strains of Lactobacillus crustrum, preferably LMG P-29154. In a particular embodiment, the baked product may be a non-leavened, yeast-leavened or chemically-leavened baked product, the main ingredient of which is flour derived from cereals. The baked product may also contain fats or fat substitutes, sugars, eggs, gluten, starch, hydrocolloids, enzymes, emulsifiers, oxidizing or reducing compounds, prebiotic compounds and/or improvers. Examples of baked products are bakery and patisserie products. Examples of bakery products are bread, steamed bread, baguettes, rolls, soft rolls, donuts, buns, microwave buns, danishes, hamburger rolls, pizza and pita bread. Examples of patisserie products are cakes, loaf cream and pound cakes, cup cream and pound cakes, sponge cakes, muffins, cake donuts, brownies and waffles. Preferably, the sourdough of the invention is used to produce steamed buns.
The invention is illustrated by the following non-limiting examples.

Example 1: Isolation and characterization of a novel strain of Lactobacillus clastrum Lactobacillus clastrum LMG P-29154 was isolated from a Chinese strain source. On a Petri dish, the Lactobacillus clastrum LMG P-29154 strain (FIG. 1A) shows morphological differences from the reference L. clastrum LMG23699 (FIG. 1B) (see FIGS. 1A and 1B).

Example 2: Liquid sourdough strains: Lactobacillus clastrum LMG P-29154 (deposited on 12 November 2015 at BCCM/LMG (Laboratorium voor Microbiologie-Bacterienverzameling; Ghent University; KLLedeganckstraat 35; B-9000, Ghent, Belgium), Lactobacillus clastrum LMG23699 obtained from the BCCM/LMG bacterial collection.

The pure strain was grown in MRS medium (peptone 10 g/l, meat extract 8 g/l, yeast extract 4 g/l, glucose 20 g/l, Tween 80 1 g/l, _{K2HP04 2 g} /l, _Na3H2O acetate 5 g/l, triammonium citrate 2 g/l, _{MgSO4.7H2O 0.2} g/l, MnSO4.4H2O 0.05 _g /l, pH 6.5) and used to inoculate a mixture of 175 g wheat flour (Pur8, Ceres, Belgium) and 325 g water. The mixture was incubated at 37° C. for 48 h in a shake flask rotating at 100 rpm.

The pH and total titratable acidity (TTA) of the sourdoughs were measured during fermentation. TTA refers to the amount of 0.1N NaOH (in ml) required to bring 10 g of product to a pH of 8.4.

Example 3: Dried sourdough Lactobacillus clastrum LMG P-29154 strain was grown in 50 ml of MRS medium (peptone 10 g/l, meat extract 8 g/l, yeast extract 4 g/l, glucose 20 g/l, Tween 80 ₁ g/l, K2HP04 ₂ g/l, _Na.3H2O acetate 5 g/l, triammonium citrate _{2 g} /l, MgSO4.7H2O _0.2 g/l, MnSO4.4H2O _0.05 g/l, pH 6.5) for 2-3 days at 35° C. 150 μl of culture from the previous step was inoculated into 1.5 L of fresh MRS medium and incubated at 35° C. for 2-3 days.

The culture was then centrifuged (4000 rpm for 10 min) and rinsed before inoculation in Fermentolevain. A blend of 10.5 kg flour and 19.5 kg water was produced in Fermentolevain (FL80-Bertrand-Puma, France). This blend was inoculated with the rinsed biomass of two 1.5 liter flasks. Fermentation took place at 35°C for 24 hours.

After the first fermentation, the mixture was further inoculated with yeast (baker's yeast Saccharomyces cerevisiae) in an amount corresponding to approximately 10 ⁷ cfu/g (colony forming units/g) and incubated at 30° C. and 100 rpm for 24 hours.

After fermentation, the pH of the sourdough was adjusted with sodium carbonate (Na ₂ CO ₃ ) to a pH of about 4-4.2. The sourdough was transferred to a mixing tank feeding a Niro P6.3 spray tower. The airflow inlet temperature was set at 185° C. with an airflow of about 630 kg/h. The airflow outlet temperature varied from 120° C. at the start to 90-100° C. at the end. The product (inlet flow rate about 151/h) was sprayed into the tower by a centrifugal pump rotating at 1.5 Hz. The dried product was collected in a cyclone located after the bottom of the spray chamber.

Example 4: Comparison of aroma by SPME-GC-MS The content of volatile molecules in the sourdoughs of Example 2 was compared by SPME-GC-MS (solid phase microextraction (SPME) and gas chromatography-mass spectrometry (GC-MS)).

The HS-SPME-GC-MS setup consisted of a gas chromatograph mass spectrometer HP7890A/5975C (Agilent Technologies™) equipped with an MPS-2W autosampler (Gerstel™). ChemSensor™ software (G1701BA, version B.01.00, Agilent Technologies™) and The unscrambler™ multivariate analysis software (9.8, Camo™, Oslo, Norway) were loaded onto the GC-MS system.

A sample weight of 1 g of each sourdough was weighed into a 20 ml glass vial, which was closed with a magnetic cap with a silicone/PTFE septum. The samples were preheated at 40°C for 10 min and volatile molecules were extracted isothermally by SPME (Fibre type DVB/CAR/PDMS Supelco Gray™ 57329-U) for 30 min. After isolation by HS-SPME, splitless thermal desorption from the sourdough fibres was carried out at 250°C for 5 min in a GC injector (SPME liner with an internal diameter of 0.75 mm). The GC column (RESTEK Stabilwax column (fused silica)) was maintained at 40°C for 7 min and then programmed to a temperature of 230°C at a rate of 16°C/min, which was held for 8 min. Helium was used as the carrier gas (1 ml/min). The injector and detector (MS-source) were maintained at 240°C and 230°C, respectively. Mass spectra were measured by electron impact at 70 eV. TIC (total ion current) chromatograms were recorded monitoring the mass-to-charge ratio (m/z) range of 40-180 amu. Identification of volatiles was performed by comparing the mass spectra of different components with those in the Mass Spectral Library Wiley275™ (J.Wiley&Sons LTD™).

The results of the analysis are shown in Figure 2. It shows that the aroma profile of the sourdoughs is different. The sourdough obtained with Lactobacillus clastrum P-29154 shows higher levels of acetic acid, isovaleric acid and benzyl alcohol, among other molecules.

Example 5: Steamed Buns Steamed buns were made using the sourdough of Example 1. Prior to being added to the recipe, the sourdough of Example 1 was neutralized with sodium carbonate (Na ₂ CO ₃ ) to a pH of about 4.0-4.2. The composition of the bun dough is shown in Table 2.

The ingredients were mixed in a Diosna mixer at low speed (110 rpm) for 5 minutes and high speed (220 rpm) for 1 minute. The dough was then proofed for 120 minutes at 37°C and 85% humidity. The dough was then mixed with 25% fresh flour and 3% water for 3 minutes at low speed and 1 minute at high speed. The dough was then pressed in a sheeter to a thickness of 5-7 mm. The sheet was rolled and cut into 50 g pieces. Each piece was then rolled by hand. The dough pieces were then placed on a tray and proofed for 25 minutes at 37°C and 85% humidity. The dough was then placed in a steamer and steamed at 100°C for 15 minutes.

Triangle test Two types of steam buns were presented to a panel of 19 people in a triangle test. Three samples were proposed in this test. Two of them were identical and the third was different. People were asked to find the sample that was different from the other two samples. It was mandatory to respond.

The number of correct answers was counted. A statistical value (p-value) was then calculated and compared to the level of risk taken for the test. A p-value lower than the risk indicates a significant difference.

11 out of 19 judges found the correct answer.

The p-value (2.41%) was inferior to the alpha risk level of the study (10%) and indicates that there was a significant perceptible sensory difference between the steamed buns produced with sourdough obtained with L. crustrum LMG P-29154 and the buns obtained with sourdough obtained with L. crustrum LMG23699.

Example 6: Northern China-type steamed breads
Northern Chinese steamed bread was made using the sourdough of Example 3. The same recipe was used to make a reference steamed bread without sourdough. The ingredients are shown in Table 3.

All ingredients were mixed in a Diosna mixer at low speed (110 rpm) for 5 minutes. The dough was then pressed to a thickness of approximately 5-7 mm. This pressing was repeated 5 times. The dough sheet was then rolled up and cut into 50 g pieces. The dough pieces were then rolled and placed on a tray. The dough was then given a final proof at 35-37°C and 85% humidity for 35 minutes. After the final proof, the dough was steamed at 100°C for 12 minutes. The steamed bread was cooled to room temperature and optionally stored frozen for 1 week.

Consumer Preference Test The steamed bread was evaluated by a consumer panel. Participants were asked to indicate their preference for one sample compared to the other samples in terms of overall quality and flavor quality. 97 participants participated in the evaluation. The results were analyzed using XLSTAT with Wilcoxon model. The results are shown in Table 4.

The results show that there was a significant difference in the participants' preference for one of the bread types (p<α), both in terms of overall quality and flavor quality. Participants preferred the steamed bread made with sourdough obtained with Lactobacillus crustrum LMG P29154.

Example 7: Southern Chinese steamed bread Southern Chinese steamed bread was made using the sourdough of Example 2. The same recipe was used to make a reference steamed bread without sourdough. The ingredients are shown in Table 5.

All ingredients were mixed at low speed (110 rpm) for 5 minutes. The dough was then pressed to a thickness of approximately 5-7 mm. This pressing was repeated 10 times. The dough sheet was then rolled up and cut into small pillow-shaped dough pieces of 20 g. The dough pieces were then proofed at 35-37°C and 85% humidity for 35 minutes. After the final proof, the dough was steamed at 100°C for 12 minutes. The steamed bread was cooled to room temperature and optionally stored frozen for 1 week.

Consumer Preference Test The steamed bread was evaluated by a consumer panel. Participants were asked to indicate their preference for one sample compared to the other samples in terms of overall quality and flavor quality. 118 participants participated in the evaluation. The results were analyzed using XLSTAT with Wilcoxon model. The results are shown in Table 6.

The results show that there was a significant difference in the participants' preference for one of the bread types (p<α), both in terms of overall quality and flavor quality. Participants preferred the steamed bread made with sourdough obtained with Lactobacillus crustrum LMG P29154.

Claims (17)

Lactobacillus clastrum strain deposited in the BCCM/LMG culture collection under accession number LMG P-29154. A baking composition for producing a bakery product comprising the strain according to claim 1. 3. The baking composition of claim 2 , wherein the baking composition is selected from a starter culture for a bakery composition, a sourdough, or a sourdough product. 4. The baking composition according to claim 2 or 3 , further comprising one or more strains of lactic acid bacteria and/or one or more strains of yeast. 5. The baking composition of claim 4 , wherein the one or more lactic acid bacteria strains are selected from acid-forming bacteria and/or the one or more yeast strains are selected from Saccharomyces, Candida, Pichia, Kluyveromyces or Hansenula strains. 6. The baking composition according to claim 4 or 5 , wherein the one or more lactic acid bacteria strains are selected from the genus Leuconostoc or Lactobacillus. 7. The baking composition according to claim 4, wherein the one or more lactic acid bacteria strains are selected from Lactobacillus plantarum, Lactobacillus brevis, Lactobacillus sanfranciscensis and/or Lactobacillus reuteri and/or the one or more yeast strains are selected from Saccharomyces cerevisiae and Saccharomyces exiguus. A baking composition according to any one of claims 2 to 7 , wherein the baking composition is a sourdough or a sourdough product. 9. The baking composition of claim 8 , wherein the baking composition further comprises grains or grain pieces, and optionally sugars and/or minerals. 10. The baking composition of claim 8 or 9 , wherein the baking composition is fermented by at least one Lactobacillus clastrum strain as defined in claim 1. 11. The baking composition according to claim 8 , wherein the baking composition is an active or inactive sourdough or sourdough product. The baking composition of claim 2 , wherein the baking composition is a starter culture. A method for producing a sourdough or a sourdough product according to any one of claims 8 to 11 , comprising the steps of:
- fermenting a (liquid) mixture comprising cereals and/or cereal pieces and liquid with at least one Lactobacillus clastrum strain LMG p-29154 to obtain a (liquid) sourdough; and - optionally drying the (liquid) sourdough to obtain a dry composition. The method of claim 13 , wherein the liquid is water. Use of a baking composition according to any one of claims 2 to 12 in the manufacture of a food product. A bakery product comprising a baking composition according to any one of claims 2 to 12 . 17. The bakery product of claim 16 which is a steamed bun.

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