PL238154B1 - Bacterial starter culture, leaven containing it, method for producing bakery products and application of the bacterial starter culture of the leaven for production of the bakery products - Google Patents

Description

Description of the invention

TECHNICAL FIELD

The subject of the invention is a bacterial starter culture containing lactic acid bacteria in the form of biomass for making sourdoughs from wholemeal or whole grain flour, including lactic acid bacteria selected for the optimal technological effect and functional properties of wholemeal or whole grain bread. The invention also relates to a sourdough containing a bacterial starter culture according to the invention, a method of producing a bread and the use of a bacterial starter culture or a sourdough according to the invention for the production of bread.

STATE OF THE ART

Consumption of wholemeal bread is recommended by human nutrition specialists as it is very rich in dietary fiber and many bioactive ingredients, which give them the character of a health-promoting food (Bartnikowska, 2009; Jurga, 2011). Such bread is also recommended in slimming diets because it is characterized by a low glycemic index (about 50), unlike white flour bread (70-95) (Gambuś and Litwinek, http://dieta.mp.pl/zasady/show .html? id = 74904). Therefore, this bread meets the WHO recommendations in the obesity program, regarding the preferences for consuming products with a glycemic index not exceeding 70. The increasing awareness of consumers forces the producers of bread to pay attention to covering the market demand for bread with functional properties, made of both traditional bread raw materials, i.e. wholemeal rye or wheat flour as well as unconventional and even relict raw materials, i.e. spelled flour (Triticum spelta). Due to the difficulties in meeting these requirements, there are generally only products available on the market with wholemeal flour, made from sourdoughs based on light flour or with baking mixes containing also many other additives, admittedly allowed, but certainly not recommended by nutritionists as pro-health products and not falling under the categories of traditional and organic bread. The production of tasty and acceptable rye bread requires acidification of the flour, while wheat bread, including spelled bread, is baked mainly with yeast, based on alcoholic fermentation. In the era of using starter cultures for the production of baker's leaven, it is recommended to use acidification of all types of flour, due to the favorable processes taking place during its fermentation: first of all, the production of organic acids that affect the taste and aroma of bread, the production of B vitamins, inactivation of pathogenic organisms, decomposition of phytates, thus increasing the digestibility of minerals, decomposing mycotoxins and extending the freshness of bread. Phytates (salts of phytic acid with various elements) found commonly in cereal grains constitute the main reserve of phosphorus, myo-inositol and minerals (Chayen, 1994; Graf and Eaton, 1990). Myo-inositol hexophosphate (ΙΡθ) in cereals accounts for 94-97% of the total phytate content (Kasim and Edwards, 1998; Pontoppidan et al., 2007). The greatest amount of phytates is found in the outer layer of the grain and in the embryo, therefore products made from wholemeal flour contain higher concentrations of these compounds than products from white, highly refined flours (Pontoppidan et al., 2007). Six reactive phosphate groups are responsible for strong complexing properties, especially in relation to positively charged molecules of proteins, amino acids and divalent metals, reducing their functionality, digestibility and absorption (Żyła, 2005). Phytates limit the use of elements such as Ca, Mg, Fe, Zn, Se as well as Cu, Co and Mn from the gastrointestinal tract (Ahn et al., 2004; Liu et al., 1998; Park et al., 2006; Sandberg et al., 1999; Zhou and Erdman, 1995). Only ΙΡθ and myo-inositol pentaphosphate (IP5) have a negative effect on the bioavailability of the listed elements (Garcia-Estepa et al., 1999; Plaami, 1997; Sandberg et al., 1989). In the digestive tract of humans and monogastric animals, ΙΡθ and IP5 also inhibit the activity of enzymes important in digestive processes, especially proteins and starch (Żyła, 2005). In the production process of bread, the phosphorolytic activity leading to the formation of lower myo-inositol and myo-inositol phosphates is due to the enzymes present in the flour, pH-appropriate enzymes generated during fermentation, as well as phytases produced by certain microorganisms (lactic acid bacteria or yeast) (De Angelis , 2003; Duliński et al., 2015). The lower myo-inositol phosphates (IP1-IP4) bind minerals only to a small extent or the complexes they create, they are more soluble in water, and thus more accessible to the human and animal organism. At the same time, the literature provides data on the possible therapeutic use of compounds released from ΙΡθ - myo-inositol and its lower phosphates (Liu et al., 1998).

PL 238 154 B1

Although many factors influence the fermentation of flour and the production of sour soups, the quality of the fermentation starter is the most important, therefore, in recent years, intensive research has been carried out with various starter compositions, i.e. appropriately selected and selected bacterial and yeast cultures that faithfully reproduce the biocenosis of sourdoughs ensuring that the technologically required characteristics of the dough are maintained, and that the final product is given the beneficial properties desired by consumers. One of the technological barriers that make it difficult to repeat the quality of bread, especially rye and flour from primary cereals, is the negligible supply of selected bacterial strains and starter cultures for sourdoughs and sour soups on the market. It should be emphasized that the currently used starter cultures were selected in terms of the correct fermentation and the related acidifying activity, possibly the production of aromas. Given the potential of lactic acid bacteria, both in terms of technological and functional characteristics, there are great opportunities to introduce new starter cultures with the desired properties. The production and variety of bakery starter cultures is very small compared to another lactic acid fermentation based food industry such as dairy.

SUMMARY OF THE INVENTION

Appropriate strains isolated from spontaneous cranes (produced on the basis of wholemeal flour) and properly composed starter cultures intended for their use in the same environment, with the participation of strains showing favorable technological and functional activities, which are the subject of the invention, constitute an advantage over the currently used and generally available starter cultures. It is well known that the fermentation process, as well as the final result, which is bread, largely depend on the flour used, climatic conditions, and the natural biodiversity of the grains. Even the best developed starter cultures developed in another region of the world do not have to work under different conditions. It should be emphasized that the lactic acid bacteria being the subject of the invention were isolated from cranes produced on the basis of Polish wholemeal flours. This is undoubtedly a great advantage compared to starters produced in other countries - adapted to work in a different environment. The isolated bacterial cultures should show the best adaptation to this type of leaven. According to the knowledge of the present inventors, no products dedicated to wholemeal bread have been introduced to the Polish market so far. Despite the presence of several types of starters on the market, there is a great need for new sets of clearly identifiable microorganisms, with undeniable advantages, allowing to enrich the aroma and taste of the bread and diversify its assortment. There is an increase in the demand for new starters for the production of wholemeal flour bread, also because the demand for various types of healthy bread made from natural ingredients is constantly increasing.

The aim of the invention was to develop the composition of the starter culture for making sourdoughs from wholemeal flour for wholemeal bread, containing lactic acid fermentation bacteria selected for the optimal technological effect and functional properties of wholemeal bread.

The strains are described:

- Lactobacillus plantarum (internally marked as 2MI8 or IBB3249) deposited with PCM (Polish Collection of Microorganisms of the Institute of Immunology and Experimental Therapy of the Polish Academy of Sciences in Wrocław) on May 5, 2017 under the number: B / 00117,

- Lactobacillus plantarum (internally marked as 6PIII6B or IBB3255) deposited in the PCM on May 5, 2017 under the number: B / 00118,

- Lactobacillus brevis (internally designated as 2MIII4 or IBB 3227) deposited with PCM on May 5, 2017 under number: B / 00119, and

- Weisell confus (internally marked as 6PI3 or IBB3282) deposited with the PCM on May 5, 2017 under the number: B / 00120.

In addition, starter cultures are presented, which include:

- Lactobacillus plantarum deposited in PCM as B / 00117 in combination with Lactobacillus plantarum deposited in PCM as B / 00118 and Lactobacillus brevis deposited in PCM as B / 00119 - Set 11, preferably with a mixture ratio of individual strains of 1: 1: 1 respectively ,

- Lactobacillus plantarum deposited as B / 00117 in combination with Lactobacillus plantarum deposited as B / 00118 and Lactobacillus brevis deposited as B / 00119 and Weisella confusa deposited as B / 00120 - Set 12, preferably with a 1: 1 ratio of the individual strains in the mixture respectively : 1: 1, or

PL 238 154 B1

- Lactobacillus plantarum deposited as B / 00117 and Weisella confusa deposited as B / 00120 - Set 12B limited to two strains, the ratio of the individual strains in the mixture preferably being 3: 1, respectively.

The invention relates to a bacterial starter culture containing a mixed culture of a lactic acid bacteria in the form of biomass containing Lactobacillus plantarum strains deposited as B / 00117 in combination with Lactobacillus plantarum deposited as B / 00118 and Lactobacillus brevis deposited as B / 00119 and Weisella confusa / 00120 deposited.

In a preferred bacterial starter culture, the ratio of Lactobacillus plantarum strains deposited as B / 00117 to Lactobacillus plantarum deposited as B / 00118 to Lactobacillus brevis deposited as B / 00119 to Weisella confus deposited as B / 00120 is 1: 1: 1: 1, respectively.

The culture according to the invention preferably contains at least 1 × 10 ¹⁰ cfu / ml of bacteria in the biomass. The invention also relates to new bacterial strains: Lactobacillus plantarum - deposited under number: B / 00117, Lactobacillus plantarum, deposited under number: B / 00118, Lactobacillus brevis, deposited under number: B / 00119 and Weisella confusa, deposited under number: B / 00120.

In a preferred bacterial starter culture, the ratio of Lactobacillus plantarum strains deposited as B / 00117 to Lactobacillus plantarum deposited as B / 00118 to Lactobacillus brevis deposited as B / 00119 to Weisella confus deposited as B / 00120 is 1: 1: 1: 1, respectively.

The culture according to the invention preferably contains at least 1 x 1010 cfu / ml of bacteria in the biomass. The invention also relates to new bacterial strains: Lactobacillus plantarum - deposited under number: B / 00117, Lactobacillus plantarum, deposited under number: B / 00118, Lactobacillus brevis, deposited under number: B / 00119 and Weisella confusa, deposited under number: B / 00120.

The invention also relates to a bacterial composition comprising at least one new bacterial strain according to the invention.

The subject of the invention is a method for the production of bread which comprises the step of adding a new bacterial strain according to the invention and / or a bacterial composition according to the invention.

The use of the new strain according to the invention and / or the bacterial composition according to the invention for the production of bread is also an object of the invention.

The invention also relates to a wholemeal or whole grain sourdough which comprises the starter culture according to the invention.

Preferably, the leaven is made of wholemeal, rye flour and is intended for the production of bread without the addition of yeast.

In a preferred sourdough, the starter culture is a bacterial starter culture comprising the lactic acid bacteria in the form of biomass according to the invention.

In another preferred embodiment, the leaven is made of wholemeal rye flour and is intended for the production of bread with yeast addition.

In another preferred embodiment of the sourdough, the starter culture comprises essentially only Lactobacillus plantarum strains deposited as B / 00117, Lactobacillus plantarum deposited as B / 00118, Lactobacillus brevis deposited as B / 00119 and Weisella confusa deposited as B / 00120 in a 1: 1 ratio: 1: 1.

In a further embodiment, a preferred leaven is spelled flour.

The invention also relates to a method of preparing a bread which comprises the step of adding a starter culture according to the invention or a sourdough according to the invention.

The invention also relates to the use of the starter culture according to the invention or the sourdough according to the invention for the production of bread.

As used herein, the term "comprises" a given bacterial strain or strains means that other, unspecified bacterial strains may also be present. The term "essentially exclusively contains" a given bacterial strain or strains means that other, unspecified bacterial strains may be present only in small amounts without significantly affecting the biological properties of the mixture of strains thus described.

In this application, the term "wholemeal flour" means flour defined as type 2000 according to the Polish Standard PN-A-74032: 2002 Cereal preparations. Rye flour.

In this application, by the term "whole grain flour" is meant a flour defined as whole grain flour, not limited to the type of flour; with an ash content from 1.21% to 2.00%, classifying

PL 238 154 B1 is a type: 1400 (sieve flour), 1850 (graham flour) or 2000 (wholemeal flour) depending on the batch of flour according to the Polish Standard: PN-A-74022: 2003 Grain preparations. Wheat flour.

In the present application, by the term "spelled flour" is meant a flour in which the spelled flour (Triticum spelta) content is 100%.

In the present application, the term "starter" is understood to mean the first phase of making the dough using a three-phase method, derived from flour and water in a ratio of 2: 3 with the addition of a starter culture.

In the present application, the term "sourdough" is understood to mean the second phase of dough management using a three-phase method obtained from one part of the starter and nine parts of flour and water in a ratio of 2: 3.

In the present application, the term "main dough" is understood to mean the third phase of dough making by the three-phase method obtained by adding the leaven to flour, water, salt and, in the case of dough for baking bread with the addition of yeast - baker's yeast and mixing. In this application, by the term "bread" is meant the fermented dough, segmented, left to rise and baked.

In this application, the term "bread" is understood to mean a general term relating to products baked with flour, water and salt, in particular breads.

SHORT DESCRIPTION OF THE FIGURES

Fig. 1 shows the acidification activity of the starter culture for making sourdoughs from wholemeal flour expressed as active acidity and titratable acidity monitored during the fermentation process (0-1 days, start; 1-2 days, leaven) and during the storage of the sourdough at 12 ° C ( 2-7 days) for wholemeal rye flour versus commercial starter culture.

Fig. 2 shows the acidification activity of the starter culture for making sourdoughs from wholemeal flour expressed as active acidity and titratable acidity monitored during the fermentation process (0-1 days, start; 1-2 days, leaven) and during the storage of the sourdough at 12 ° C ( 2-7 days) for wholegrain spelled flour.

Fig. 3 shows the number of microorganisms in samples of wholemeal rye sourdoughs produced with the starter culture relative to the commercial starter culture and the spontaneous sourdough after 72 h of fermentation.

Fig. 4 shows the number of microorganisms in samples of sourdoughs from whole grain spelled flour produced with the starter culture versus spontaneous sourdough after 72 h of fermentation.

Fig. 5 shows the taxonomic structure of bacteria at the level of the order of Lactobacillales in the wholemeal rye sourdough produced with Set 12 of the starter culture and in the spontaneous rye sourdough after 72 h of fermentation. The results constituting a minimum of 0.5% of all readings assigned to the order of Lactobacillales were included.

Fig. 6 shows an image of the electrophoretic separation of RAPD reaction products using the RAPD-B10 and RAPD-GACA primers for the strains included in the starter culture: 1 - B / 00117, 2 - B / 00118, 3 - B / 00119, 4 - B / 00120; M - DNA size marker.

Strains of lactic acid bacteria for use in the starter culture according to the invention were isolated from cranes produced exclusively from wholemeal rye flour and selected from a group of several dozen newly isolated strains. Strains were identified by species on the basis of API 50CH tests, by sequencing of 16S rRNA-encoding genes and by mass spectroscopy (MALDI TOF MS). The obtained nucleotide sequences showed a consistency of 99% or 100% with the sequences of the appropriate species deposited in the GenBank database. Differentiation of isolates within species was performed using the RAPD-PCR method. Strains representing the dominant species found in cranes were selected for a broad spectrum of sugar catabolism, the ability to produce exopolysaccharides and antimicrobial properties, with particular emphasis on activity against bacillus spore bacteria. The Lactobacillus plantarum IBB3249 strain deposited as B / 00117 is capable of catabolizing 21 sugars out of the 49 tested sugars, while Lactobacillus plantarum IBB3255 deposited as B / 00118 ferments 20 sugars. Lactobacillus brevis IBB 3227 strain deposited as B / 00119 is active against Brevibacillus brevis spore bacteria. The Weisell confus strain IBB3282 deposited as B / 00120 produces the exopolysaccharide-dextran (gluco-oligosaccharide - GOS) on a sucrose-containing medium. All strains are capable of breaking down maltose.

The selected bacterial strains according to the description in Example 1 in the form of mixed cultures obtained as in Example 2 were used to prepare sourdoughs from wholemeal rye flour and compared to a commercial culture intended for acidification of rye flour. Kits of the cultures wiped off

The fermenters were tested for optimal sourdough fermentation. The bacterial starter culture of the invention has optimal acidifying activity, and the resulting sourdoughs remain stable for 5 days until the end of the monitoring period as shown in Example 3 and Fig. 1.

The starter culture was also tested on spelled wheat flour, obtaining correspondingly acidified and stable sourdoughs as shown in Example 4 (Fig. 2).

The sourdoughs obtained in Example 3 on wholemeal rye flour were used to produce wholemeal rye bread without (Example 5) and with the addition of yeast (Example 6), which was compared with the bread produced using a commercial culture intended for acidification of rye flour and with bread produced on the basis of spontaneous leaven. . The selected sets of starter cultures were also tested for the ability to break down phytates, reducing the bioavailability of micro- and macroelements, both for the microflora of sourdoughs, as well as for people - bread consumers, and for the possibility of obtaining high-quality bread made with their participation. In addition, the sourdoughs obtained on wholegrain spelled flour (Example 4) were used to produce spelled bread with the addition of yeast (Example 7).

For all sourdoughs obtained in Examples 3 and 4, microbiological analysis was performed using the culture dependent methods shown for sourdoughs on wholemeal rye flour in Example 8 (Fig. 3) and for sourdoughs on wholegrain spelled flour in Example 9 (Fig. 4), respectively. The quantification of aerobic and anaerobic bacteria as well as thermophilic and mesophilic lactic bacteria show that these values are similar in spontaneous and supplemented cranes. The number of mesophilic lactic acid bacteria was similar to that for spontaneous leaven, approx. 10 ⁹ cfu / ml. In all samples the number ranged from 5 x 10 ⁸ to 9.6 x 10 ⁹ cfu / ml. As in the case of spontaneous sourdoughs, the number of yeasts was lower by 2-3 logarithmic orders compared to the number of mesophilic lactic bacteria. No mold was grown for any of the cranes samples. Moreover, it was noticed that the use of some starter culture kits significantly reduced the number of spore-forming bacteria (to the level of approx. 102 cfu / ml) compared to spontaneous cranes where the number was approx. 104 cfu / ml. The inhibition of the growth of spore-forming bacteria by the use of the inventive starter culture was comparable to the results obtained with the commercial starter culture.

In addition, bacterial diversity analysis was performed using culture-independent methods for the sourdough obtained in Example 3 on wholemeal rye flour using Set 12 of the starter culture (Example 10). The results are presented against spontaneous rye leaven after 72 h of fermentation (Fig. 5). The results obtained as a result of bioinformatics analysis show that the sourdough environment is dominated by bacteria of the Lactobacillus genus. The next most numerous groups were unclassified Lactobacillales and the genus Weissella.

The culture-dependent and culture-independent microbiological analysis shows a high similarity of the leaven obtained with the starter culture constituting the subject of the invention to the spontaneous control leaven or prepared on the basis of the commercial starter culture. The obtained results confirm the legitimacy of using such a starter culture for the fermentation of flour in order to produce leaven with an appropriate composition of microorganisms.

The aim of the invention has been achieved because the developed starter culture consisting of lactic acid bacteria allowed to obtain a suitable biodiversity, good souring activity, stable sourdoughs from wholemeal rye flour or wholegrain spelled wheat. With the use of sourdoughs based on the starter culture, which is the subject of the invention, bread is obtained of a similar or better quality than bread produced with the use of the comparable commercial culture or spontaneous sourdough. Moreover, it has been shown that wholemeal rye bread produced thanks to the use of starter culture is characterized by a low content of myo-inositol phosphates, in particular higher forms (IP6 and IP5), which increases the bioavailability of micro and macro elements present in bread. Produced as a result of the activity of phytases: myo-inositol and its lower phosphates are biostimulating compounds with pro-health properties.

The invention is illustrated by the following non-limiting examples.

EXAMPLES

P r z k ł a d 1

During the microbiological analysis of spontaneous cranes based on wholemeal rye flour, the isolation of lactic acid bacteria was carried out in order to create a collection of microorganisms accumulating, among others, bacteria with potentially high adaptation to the environment of cranes based on wholemeal flours. Bacteria were isolated from MRS, PCM and Blickfeldt solid media. The morphology of all isolates was assessed macroscopically and microscopically. A collection of 90 potentially high isolates was created

The adaptation to the environment of cranes based on wholemeal flour. Bacteria from the created collection were identified by sequencing of genes encoding 16S rRNA. Their species affiliation was determined. For this purpose, the genomic DNA of individual isolates was isolated using enzymatic lysis (lysozyme and mutanolysin) using ready-made genomic DNA isolation kits by A&A Biotechnology (Poland). Then, the genes encoding 16S rRNA were amplified on the template of the isolated DNA with the use of primers specific for specific bacteria. The products of the PCR reaction were purified and submitted for sequencing in the Laboratory of DNA Sequencing and Oligonucleotide Synthesis of IBB PAN. The obtained nucleotide sequences were prepared for further analysis using the Clone Manager program. The obtained nucleotide sequences were compared with the nucleotide sequence databases using the BLAST program. For selected groups of microorganisms belonging to the genera: Lactobacillus, Pediococcus, Lactococcus, Leuconostoc and Weissella, isolates were differentiated within species using the method of random amplification of polymorphic DNA fragments (RAPD). RAPD analysis was performed on the template of the isolated DNA in duplicate for each of the two types of primers RAPD-GACA (SEQ ID NO. 1: GACAGACAGACAGACA) and RAPD-B10 (SEQ ID NO. 2: CTGCTGGGAC). The amplification products were separated on agarose gels, visualized under UV light and photographed. The obtained images were analyzed with the use of Syngen software that allows to compare the profiles of the obtained bands. The conducted analysis allowed for the differentiation of isolates belonging to a specific species, and thus the identification of strains characterized by a unique genotype. The image of the electrophoretic separation of RAPD reaction products using the RAPD-B10 and RAPD-GACA primers for the strains included in the starter culture according to the invention is shown in Fig. 6. The species affiliation of the strains included in the starter culture according to the invention was confirmed by mass spectroscopy using the MALDI apparatus. TOF when making a deposit to the PCM. The collected bacterial strains were characterized in terms of physiological and biochemical properties. The bacteria were tested for amylolytic properties, the ability to produce and secrete exopolysaccharides, acidification of the medium, production of aroma-forming compounds and antimicrobial properties. All bacteria with amylolytic properties have been identified to the genus Enterococcus. Among the isolates producing exopolysaccharides on the substrate with sucrose (as the only carbon source) were bacteria belonging to the genera Weissella and Leuconostoc.

Among the acidifying bacteria isolated from the Blickfeldt medium, bacteria belonging to the genera Lactococcus and Enterococcus were selected. In addition, more bacteria with acidifying potential were isolated from other media, i.e. bacteria of the genus Lactobacillus, for which the Blickfeldt medium was not an optimal medium. Among the isolates with the ability to metabolize citrate salt to aroma-forming compounds (diacetyl, acetoin), only bacteria belonging to the genus Enterococcus were identified. The antagonistic properties for rye flour isolates for which the antagonistic activity against spore bacteria from the genera Bacillus, Brevibacillus and Lysinibacillus were found. For selected isolates obtained from cranes, metabolic profiles were tested using API 50 CH kits. The strains included in the starter culture are characterized by the ability to catabolize the following sugars:

B / 00117 (L-arabinose, D-ribose, D-galactose, D-glucose, D-fructose, D-mannose, D-mannitol, N-acetyl-glucosamine, Amygdalin, Arbutin, Asculin, Salicin, D-cellobiose, D-maltose, D-lactose, D-melibiosis, D-sucrose, D-trehalose, D-melesitose, D-raffinose, β-gentiobiose);

B / 00118 (D-ribose, D-galactose, D-glucose, D-fructose, D-mannose, D-mannitol, D-sorbitol, N-acetyl-glucosamine, Amygdalin, Arbutin, Asculin, Salicin, D-cellobiose, D-maltose, D-lactose, D-melibiosis, D-sucrose, D-trehalose, D-raffinose, β-gentiobiose);

B / 00119 (L-arabinose, D-ribose, D-xylose, Methyl-β-D-xylopyranoside, D-galactose, D-glucose, D-fructose, Amygdalin, Aesculin, D-maltose, D-melibioside);

B / 00120 (D-ribose, D-xylose, D-galactose, D-glucose, D-fructose, D-mannose, N-acetyl-glucosamine, Amygdalin, Arbutin, Aesculin, Salicin, D-cellobiose, D-maltose, D-sucrose, β-gentiobiose, Gluconate, 2-Keto gluconate).

For isolated strains having favorable technological properties and / or a broad spectrum of catabolic abilities, coexistence tests were carried out, enabling the selection of non-mutually exclusive strains that could be included in the multi-strain starter cultures.

On the basis of the phenotypic characteristics of the strains, sets of lactic bacteria were developed for the test starter cultures.

PL 238 154 B1

P r z k ł a d 2

A starter culture for the production of sourdough from wholemeal flour, consisting of lactic acid bacteria, was prepared using a 2-liter glass fermenter Biostat B plus, manufactured by Sartorius. The biomass of each strain was obtained separately by inoculating the culture medium with MRS 3% (10% for Weisell confus) inoculum of an overnight culture obtained from a single colony. All fermentation processes were carried out at the temperature of 37 ° C; for Lactobacillus, the fed-batch system with supplementation of the carbon source (3 g glucose / h) was used for 16 h (from 12 to 28 h). The multiplication of the bacterial biomass was carried out without oxygenation and aeration. The pH was monitored and controlled at 6.5 using a 10M NaOH solution. The process was carried out for 30 hours for Lactobacillus bacteria and 7 hours for Weisell.

In order to concentrate the biomass, the output from each fermentor was centrifuged at 4 ° C, at 6,000 m3. rpm for 15 minutes, then washed with 0.9% NaCl solution and centrifuged again. The obtained pellet was suspended in a solution with cryoprotective properties (0.9% NaCl, 20% trehalose) and frozen in liquid nitrogen. Bacteria aliquots were stored at -80 ° C.

The number of bacteria in the frozen samples was determined by plating 10-fold dilutions in saline on a solid medium and determining the number of bacteria forming colonies (cfu / ml).

Then the biomass of bacterial strains was mixed:

Lactobacillus plantarum deposited as B / 00117 with strains of Lactobacillus plantarum deposited as B / 00118 and Lactobacillus brevis deposited as B / 00119 in a 1: 1: 1 ratio - Set 11;

Lactobacillus plantarum deposited as B / 00117, Lactobacillus plantarum deposited as B / 00118, Lactobacillus brevis deposited as B / 00119 and Weisella confusa deposited as B / 00120 in a ratio of 1: 1: 1: 1 - Set 12;

and Lactobacillus plantarum deposited as B / 00117 from Weisella confusa deposited as B / 00120 in a ratio of 3: 1 - Set 12B.

The above kits were used in the following examples.

The number of bacteria in the frozen samples was determined again by plating 10-fold dilutions in saline onto a solid medium and determining the number of bacteria forming colonies (cfu / ml). The preparations with the bacteria content of ¹⁰ cfu / ml were obtained.

P r z k ł a d 3

The starter culture obtained in Example 2 was used to make sourdoughs from wholemeal rye flour. First, the starter was derived from the flour and water with the addition of a single starter culture preparation, which was then used to make the leaven according to the following recipe:

Recipe to prepare 100 kg of leaven

- type 2000 rye flour 40 kg (including 4 kg used for the start)

- 60 kg water (including 6 kg used for the start), and

- commercial starter culture used for the beginning (starter culture for acidification of LV2 rye flour (bacterial-yeast) by Lesaffre, containing Saccharomyces chevalieri, Lactobacillus brevis - data from Lesaffre Bio-Corporation SA, Łódź, based on Kawka and Górecka, 2010) 20 g

[> 10 ⁸ cfu / g of product] - 2 x 10 ⁹ cfu of bacteria

[> 10 ⁸ cfu / g of product] - 2 x 10 ⁹ cfu of yeast, or

- the preparation of the starter culture according to the invention used for the start, 2 ml [10 ¹⁰ CFU of bacteria].

After the ingredients were mixed (5 min), the buds were fermented for 24 h at 30 ° C. Ripe starters were mixed with flour and water in appropriate proportions to obtain 100 kg of ready-made leaven and fermented at 30 ° C for 24 hours. The sourdoughs were then cooled and stored at 12 ° C for 5 days.

During the fermentation and during the storage of the leaven, the acidification process was monitored using a computerized system for the evaluation of acidifying activity (iCINAC). Measurements of active acidity were made every 10 minutes using the iCINAC apparatus, while the titratable acidity of sourdoughs was determined several times during the tests for a period of 7 days with the use of sodium hydroxide in the presence of phenolphthalein according to "Pieczywo - Research methods", PN-A-74108. The measurement results are shown in Fig. 1. All three starter culture preparations, Set 11, 12 and 12B described in Example 2, achieved an optimal acidification activity similar to that of the compared commercial culture, and the resulting sourdoughs remained stable for 5 days until the end of the period. monitoring.

PL 238 154 B1

P r x l a d 4

In an analogous manner as in Example 3, the starter culture obtained in Example 2 was used to produce sourdoughs from wholegrain spelled flour. Recipe to prepare 100 kg of leaven

- whole grain spelled flour 40 kg (including 4 kg used for the start)

- 60 kg water (including 6 kg used for the start), and

- a commercial starter culture used for the start (starter culture for acidification of LV2 rye (bacterial-yeast) flour from Lesaffre) 20 g, or

- the preparation of the starter culture according to the invention used for the start, 2 ml [10 ¹⁰ CFU of bacteria].

The sourness activity results for Set 12 and Set 12B for spelled flour are shown in Fig. 2. As with wholemeal rye flour, optimal sour activity was obtained and the resulting sourdoughs remained stable for 5 days until the end of the monitoring period.

P r z k ł a d 5

The sourdoughs obtained in Example 3 were used to produce wholemeal rye bread without the addition of yeast, which was compared with bread produced with the use of a commercial starter culture (starter culture for acidification of LV2 rye flour (LV2 by Lesaffre) intended for acidification of rye flour and bread produced from bread. based on spontaneous sourdoughs.

Recipe for the preparation of bread (breads weighing 0.5 kg)

Preparation of raw materials and their bearing sizes (100 kg of dough):

- type 2000 rye flour 50.6 kg

- sourdough from rye flour type 2000 17.1 kg

- salt 1.17 kg

- water (temperature 38 ° C) 3.1 kg

Production and preparation of proper dough

- mixing time - idle 7 min.

- mixing time - fast rotation 3 min.

- proper dough temperature up to 35 ° C

Dividing, forming, shaping the proper dough

- mass of a billet 0.6 kg

- time of dividing and shaping the dough into pieces 45 min.

- fermentation time of proper dough in bites up to 240 min.

The baking process

- baking temperature: 200 ° C

- baking time 60 min.

Sample names:

• VSZ1 - wholemeal rye bread exclusively with spontaneous leaven - 1 repetition • VSZ2 - wholemeal rye bread exclusively with spontaneous leaven - 2 repetition • VKZ K - wholemeal rye bread exclusively with sourdough with commercial starter culture K • VKZ 11 - wholemeal rye bread exclusively for sourdough with starter culture - Set 11 • VKZ 12 - wholemeal rye bread exclusively with sourdough with starter culture - Set 12 • VKZ 12B - wholemeal rye bread exclusively with sourdough with starter culture - Set 12B.

The obtained bread was subjected to organoleptic evaluation according to PN-A-74108: 1996, the analysis was carried out using the point method by a panel of at least 15 people with proven sensory sensitivity (Table 1). Each type of bread was classified to the appropriate quality class in accordance with PN-A-74108: 1996, adding 8 points to the sum of points from the organoleptic evaluation of each tested bread for biochemical indicators (Table 2). The weight of cold loaves was also measured, which together with the recipe were used to calculate the dough efficiency and total baking loss (Jakubczyk and Haber [1981]). The volume of the loaves was also analyzed in a Volscan Profiler (Stable Micro System, UK) and a volume of 100 g of bread was calculated based on the weight of the loaves (Table 2). Crumb moisture analysis was also performed using the drying method (according to AOAC - method No. 925.10) and the texture profile of the bread crumb using the TA.XT Plus texture analyzer (Stable Micro Systems, UK) (Table 3). Using the Exponent v. 4.0.13.0 software. and a standard macro program for the TPA test (Stable Micro Systems, UK), the hardness was calculated

PL238 154 Β1 (maximum force achieved during the first compression), resilience (the ability of the crumb to regain its original height in the time between the end of the first compression and the beginning of the second compression), cohesion (the degree of disintegration of the material under the influence of mechanical impact on the product, expressed as the ratio of the surface area under the curve of the second compression to the area under the curve of the first compression), chewiness (energy needed to crush (chew) a solid product into a swallow state - this is the product of hardness, cohesion and elasticity) and resilience - immediate elasticity of the crumb ( ability to return to the original form of a deformed sample expressed as a derivative of time by force).

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Table 2. Quality of rye bread made without the addition of yeast with fermented leaven with starter cultures

Sample name Loaf weight [g] Loaf volume [cm ³ ] Volume 100 g of bread [cm ³ ] Organoleptic assessment Crumb acidity [° kw] Average sum of points Quality class VSZ1 514b +5 897 d ± 24 175e ± 4 33.8 a ± 3.6 II 10 VSZ2 519 bc ± 7 863 c ± 21 166 d ± 4 35.8 ab ± 2.1 AND 11 VKZK 523 c ± 5 781 a ± 14 149 a +3 35.9 ab ± 1.9 AND 9 VKZ 11 523 C. ± 5 815b ± 11 156 b ± 3 36.3 b ± 2.1 AND 8 VKZ 12 518 bc ± 4 817b ± 13 158bc ± 2 35.4 ab ± 2.0 II 10 VKZ 12B 508 a ± 6 814b ± 9 160c ± 2 37.6 b ± 1.0 AND 8

The table shows mean values ± standard deviations, a, b ... - mean values marked with different letters in the column differ statistically significantly at p <0.05

The obtained Breads were stored for 7 days, and in order to determine the changes occurring during the aging of the bread, the analysis of crumb moisture was performed using the drying method (according to AOAC - method No. 925.10) and the texture profile of the bread crumb, texture analyzer ΤΑ.ΧΤ Plus (Stable Micro Systems, UK) . The results of the analysis after 5 and 7 days of storage are presented in Tables 4 and 5, respectively.

For the produced bread, the microbiological stability was also determined using the thermostat method (according to PN-A-74102: 1999) (Table 6). A randomly drawn sample of the product was thermostated at 30 ° C and possible organoleptic changes caused by molds were observed, equally the sample was also thermostated at 37 ° C in order to determine the organoleptic changes caused by aerobic amylolytic spore bacteria. All Breads were stored for a minimum of 7 days, and readings were taken after 1, 5 and 7 days of storage, and even longer until the first changes caused by microorganisms or the need to free space in the incubator for new samples. In addition to determining the microbiological stability of bread with the thermostat method, microbiological cultures were also carried out to determine in 1 g of bread: the number of aerobic amylolytic bacteria (according to PN-A-74134-4: 1998) (OLBA), the number of aerobic amylolytic bacteria (according to PN-A-74134-4: 1998) (according to from PN-A-74134-4: 1998) (OLBAP), yeast and mold numbers (according to PN-A-74134-6: 1998) (OLG) (Table 7). All microbiological analyzes were performed in triplicate on 1.5 and 7 days of bread storage.

The content of myo-inositol phosphates was determined in the obtained bread and flour (according to Chen and Li, 2003) (Table 8). In addition, in Chleby the acidity of the crumb was determined by the titration method according to PN-A-74108: 1996 and the content of substances that shape the aroma of the bread by the HPLC / UV method, based on the research of Wiseblatt and Zoumut (1963), Johnson and Linko (1964 and Lefebvre et al. ( 2002) (Table 9) Among aromatic compounds, the content of organic acids (lactic acid, acetic acid, propionic acid, citric acid, tartaric acid, malic acid and citric acid) was determined.

All analyzes of the quality, texture profiles and chemical composition of the bread were carried out in at least two replications, the final result was presented as mean values ± SD (standard deviation). One-way analysis of variance (ANOVA) was used in the Statistica 10 program. The significance of differences between the mean values was verified by Duncan's test at a = 0.05.

Wholemeal rye bread without the addition of yeast obtained from sourdough with the starter culture according to the invention for all the combinations of microorganisms proposed in the invention is characterized by a greater volume and moisture of the crumb, significantly lower hardness and chewiness of the crumb compared to the control bread produced on the basis of a commercial culture. The sourdough bread supplemented with the developed starter culture shows the same microbiological stability (6 days) as the control bread with the commercial culture.

The best organoleptic evaluation, among wholemeal rye breads without the addition of yeast, was achieved by the bread produced with the use of Set 12B. Sets 11 and 12B were rated significantly better both against spontaneous sourdoughs and against commercial culture.

Due to the best technological parameters obtained, Sets 11 and 12B are especially recommended for wholemeal rye bread without the addition of yeast.

PL238 154 Β1

The use of leaven made from wholemeal rye flour with the participation of starter culture for the production of bread, resulted in a significant reduction in the amount of myo-inositol phosphate (IP) in all rye breads without the addition of yeast, in relation to the analyzed raw materials, i.e. wholemeal flour used for baking, for which the total IP content in% DM was 1.18. The reduction in the content of these compounds was from 1.5 to 4.7 times.

PL238 154B1

Table 5. Crumb texture profile after 7 days of storage of rye bread produced without the addition of yeast with fermented leaven with starter cultures

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PL238 154 Β1

Table 7. Number of aerobic amylolytic bacteria (OLBA), aerobic spore amylolytic bacteria (OLBAP) and the number of mold and yeast (OLG) after 1, 5 and 7 days of storage of rye bread produced without the addition of yeast fermented with starter cultures

Sample name OLBA OLBAP OLG 1 5 7 1 5 7 1 5 7 [CFU / g of bread] VSZ1 0 0 0 0 0 0 2.0x10 ¹ 1.0x10 ¹ 2.0x10 ¹ VSZ2 0 0 0 0 0 0 0 0 6.0x10 ¹ VKZK 0 0 0 0 0 0 0 0 0 VKZ11 0 0 0 0 0 0 0 2x10 ¹ 2.5x10 ³ VKZ12 0 0 0 0 0 0 0 0 0 VKZ12B 0 0 0 0 0 0 0 2.9x10 ² 1.3x10 ³

Table 8. The content of myo-inositol phosphates in flour and rye bread produced without the addition of yeast with fermented leaven with starter cultures

Sample name Myo-inositol phosphate content (% m.m.) Myo-inositol phosphate content (% DM) IP6 Sum of IP IP6 Sum of IP VSZ1 0.01a ± 0.01 0.06a ± 0.00 0.02a ± 0.01 0.10a ± 0.01 VSZ2 0.02a ± 0.00 0.07a ± 0.01 0.04a ± 0.00 0.11a ± 0.01 VKZK 0.06c ± 0.00 0.25d ± 0.00 0.10c ± 0.00 0.44c ± 0.00 VKZ 11 0.04b ± 0.00 0.17c ± 0.00 0.07b ± 0.00 0.28b ± 0.00 VKZ 12 0.01a ± 0.00 0.13b ± 0.00 0.03a ± 0.00 0.25b ± 0.02 VKZ 12B 0.14d ± 0.00 0.41e ± 0.02 0.28d ± 0.02 0.80d ± 0.08

The table shows mean values ± standard deviations, a, b ... - mean values marked with different letters in the column differ statistically significantly at p <0.05

PL238 154B1

PL 238 154 B1

P r z k ł a d 6

The sourdoughs obtained in Example 3 were also used to prepare wholemeal rye bread with the addition of S. cerevisiae baker's yeast, which was compared to bread with the addition of the same baker's yeast produced with the use of a commercial starter culture intended for acidification of rye flour (starter culture for acidification of rye flour (bacterial - yeast) LV2 by Lesaffre) and with bread produced on the basis of spontaneous sourdoughs.

Recipe for the preparation of bread (breads weighing 0.5 kg)

Preparation of raw materials and their bearing sizes (100 kg of dough):

- type 2000 rye flour 50.3 kg - type 2000 rye sourdough 17.0 kg - yeast 0.55 kg - salt 1.17 kg - water (temperature 38 ° C) 31.0 kg Production and preparation of proper dough - mixing time - slow revolutions 7 min. - mixing time - fast rotation 3 min. - dough temperature up to 35 ° C Dividing, forming, shaping the proper dough - weight of the billet 0.6 kg - time of dividing and shaping the dough into bites 45 min. - fermentation time of the proper dough in the billets up to 90 minutes The baking process - baking temperature: 200 ° C - baking time 60 min.

Sample names:

• VSZ1 - wholemeal rye bread exclusively with spontaneous leaven - 1 repetition • VSZ2 - wholemeal rye bread exclusively with spontaneous leaven - 2 repetition • VKDZ K - wholemeal rye sourdough bread with commercial starter culture K and yeast • VKDZ 11 - wholemeal rye bread sourdough with starter culture (Set 11) and yeast • VKDZ 12 - wholemeal rye sourdough bread with starter culture (Set 12) and yeast • VKDZ 12B - wholemeal rye sourdough bread with starter culture (Set 12B) and yeast.

Quality, texture profile, microbiological and chemical composition analyzes of the breads were performed as described in Example 5. The evaluation results of the bread produced are shown in Tables 10-16.

PL238 154B1

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PL238 154 Β1

Table 11. The quality of rye bread made with the addition of yeast fermented with starter cultures

Sample name Loaf weight [g] Loaf volume [cm ³ ] Volume 100 g of bread [cm ³ ] Organoleptic assessment Crumb acidity [° kw] Average sum of points Quality class VSZ1 514b ± 5 897 b ± 24 175 b ± 4 33.8 a ± 3.6 II 10 VSZ2 519 b ± 7 863 a ± 21 166 a ± 4 35.8 a ± 2.1 AND 11 VKDZ K 514b ± 5 849 a ± 12 165 a ± 3 35.8 a ± 3.4 AND 5 VKDZ11 517 b ± 7 847 a ± 17 164 a ± 3 36.0 ab ± 2.2 AND 5 VKDZ 12 514b ± 6 853 a ± 11 166 a ± 3 38.1 b ± 1.6 AND 6 VKDZ12B 506 a ± 8 886 b ± 10 175 b ± 2 37.8 b ± 0.8 AND 5

The table shows mean values ± standard deviations, a, b ... - mean values marked with different letters in the column differ statistically significantly at p <0.05

Wholemeal rye bread with the addition of yeast obtained from sourdough with the starter culture for the microbial set 12 and 12B is characterized by a greater volume and moisture of the crumb, significantly lower hardness and chewiness of the crumb compared to the control bread produced on the basis of a commercial culture. The sourdough bread supplemented with the developed starter culture shows the same microbiological stability (3 days) as the control bread with the commercial culture.

The highest organoleptic evaluation among wholemeal rye breads with the addition of yeast was achieved by bread produced with the participation of Set 12. Sets 12 and 12B were evaluated significantly better both in relation to spontaneous leaven and in relation to commercial culture.

Due to the best technological parameters obtained, Sets 12 and 12B are especially recommended for wholemeal rye bread with the addition of yeast.

Fermentation of sourdoughs made of wholemeal rye flour with the participation of a starter culture resulted in a significant reduction in the amount of myo-inositol phosphate (IP) in all rye breads with the addition of yeast, in relation to the analyzed raw materials, i.e. wholemeal flour, used for baking, for which the total IP content in% sm was 1.18. The reduction in the content of these compounds was 1.7 to 2.4 times.

PL238 154B1

Table 12. Crumb texture profile on the day of baking rye bread with the addition of yeast on fermented leaven with starter cultures

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Resilience SD 0.003 0.005 o. o © 0.006 000Ό OJ Fr. about mean 0.086 c 0.073 b 0.075 b 0.091 c 0.073 b 0.064 a Chewing SD 0.94 0.59 2.13 0.04 1.08 0.34 mean 5.31 ab 4.60 a O © co o 14.56 d 10.63 c 7.58 b Cohesion SD 0.024 Ζ00Ό 6ί0Ό ζεοΌ 0.028 0.013 mean 0.295 a 0.276 a 0.268 a 0.317 a 0.298 a 0.269 a Elasticity SD 0.043 0.047 0.037 0.107 0.036 0.016 mean 0.864 a 0.901 a 0.924 a 0.874 a 0.977 a 0.943 a Hardness SD 0.95 0.94 4.03 0.52 1.10 εε'ο mean 20.74 a 18.45 a 41.69 d 52.89 e 36.51 c 29.92 b Humidity SD 0.11 0.09 CO o about 0.09 90Ό in o mean 49.07 c 49.20 c 47.96 a 48.31 b 49.25 C 50.78 d Sample name tZSA CM N > N o VKDZ11 VKDZ12 VKDZ 12B

The table shows the mean values with standard deviations (SD), a, b - mean values marked with different letters in the column differ statistically significantly at p <0.05

PL238 154 B1

Table 14. Crumb texture profile after 7 days of storage of rye bread made with the addition of yeast fermented leaven with starter cultures

PL238 154B1

Table 16. Number of aerobic amylolytic bacteria (OLBA), aerobic spore amylolytic bacteria (OLBAP) and the number of mold and yeast (OLG) after 1, 5 and 7 days of storage of rye bread made with yeast fermented with starter cultures

Sample name OLBA OLBAP OLG 1 5 7 1 5 7 1 5 7 [CFU / g of bread] VSZ1 0 0 0 0 0 0 2.0x10 ¹ 1.0x10 ¹ 2.0x10 ¹ VSZ2 0 0 0 0 0 0 0 0 6.0x10 ¹ VKDZ K 0 0 0 0 0 0 1.5x10 ¹ 4.1x10 ³ 1.4x10 ⁵ VKDZ11 0 0 0 0 0 0 0 3x10 ² 1.2X10® VKDZ 12 0 0 0 0 0 0 10 6.2x10 ³ 2.2x10 + VKDZ 12B 0 0 0 0 0 0 0 1.2x10 + 2.5x10 ^s

Table 17. Content of myo-inositol phosphates in flour and rye bread with fermented leaven with starter cultures with yeast

Sample name Myo-inositol phosphate content (% m.m.) Myo-inositol phosphate content (% DM) IP6 Sum of IP IP6 Sum of IP VSZ1 0.01 a ± 0.01 0.06a ± 0.00 0.02a ± 0.01 0.10a ± 0.01 VSZ2 0.02a ± 0.00 0.07a ± 0.01 0.04a ± 0.00 0.11a ± 0.01 VKDZ K 0.20c ± 0.01 0.39c ± 0.00 0.37c ± 0.02 0.69c ± 0.01 VKDZ 11 0.20c ± 0.02 0.39c ± 0.02 0.33c ± 0.05 0.63c ± 0.08 VKDZ 12 0.11b ± 0.00 0.27b ± 0.00 0.20b ± 0.01 0.50b ± 0.03 VKDZ 12B 0.25d ± 0.01 0.37c ± 0.00 0.44d ± 0.01 0.64c ± 0.01

The table shows mean values ± standard deviations, a, b ... - mean values marked with different letters in the column differ statistically significantly at p <0.05

PL238 154 Β1

PL 238 154 B1

P r z k ł a d 7

The sourdoughs obtained in Example 4 were also used to produce wholegrain spelled bread with the addition of yeast, which was compared with the bread produced on the basis of spontaneous sourdoughs.

Recipe for the preparation of bread (breads weighing 0.5 kg)

Preparation of raw materials and their bearing sizes (100 kg of dough):

- wholemeal spelled flour 50.7 kg - sourdough made of wholegrain spelled flour 12.7 kg - yeast 0.07 kg - salt 0.97 kg - water (temperature 38 ° C) 35.5 kg Production and preparation of proper dough - mixing time - slow speed 6 min. - mixing time - fast rotation 8 min. Dividing, shaping, shaping the proper dough - the mass of a bite 0.6 kg - time of dividing and shaping the dough into bites 45 min. - fermentation time of the proper dough in the billets up to 90 minutes The baking process - baking temperature: 200 ° C - baking time 55-60 min

Sample names:

• VSO1 - wholemeal spelled bread exclusively with spontaneous sourdough - 1 repetition • VSO2 - wholemeal spelled bread exclusively with spontaneous sourdough - 2 repetitions • VKDO 12 - wholemeal spelled bread with sourdough with starter culture (Set 12) and yeast • VKDO 12B - spelled bread whole grain sourdough with starter culture (Set 12B) and yeast

Quality, texture profile, microbiological and chemical composition analyzes of the breads were performed as described in Example 5. The evaluation results of the bread produced are shown in Tables 19-25.

PL238 154 Β1 □

D Ε φ ; poppy ^SD 9'0 0.5 0.5 Φ 'Ν Ό □ Ν Smell and ί mean LD 4.6 5.3 Lf> Ε § oh kishu o ω 9'0 9'0 0.4 0.5 ό C φ Ε φ Μ— Stay; features of me mean 2.5 2.2 2.9 cm Φ 'ω co • 0 • W _z o S SD b 8'0 0.5 0.8 Ν εΰ C >> • Ν 0 Ν Ο Porous crumb; mean CM 2.2 OJ 2.6 Elasticity of the crumb about 9'0 0.5 0.5 T- Ε, φ σ ο ό Ν _C Ο Ο ο mean 3.3 3.5 3.6 3.4 Other features of the skin o ω 9'0 0.5 9'0 0.4 mean 3.3 3.5 3.6 3.8 rdowych (Si JZ ο -g Q ω 0.4 0.4 0.0 0.6 tanda § Ν Φ lid Ο Thickness p mean 3.9 3.8 4.0 3.5 lylings p _Ω Φ Q ω (D 0.4 0.0 0.5 with dec JZ Ο cc C Ν Color sk mean 2.3 2.8 3.0 2.5 medium ones; ο. ω ο ΕΣ as 9'0 b 0.3 0.5 • 0 O and 05 0) Ο 05 C φ They look outside mean 4.5 4.8 4.9 o o and ο ώ Γ m Φ η C0 1- starter Sample name VSO1 VSO 2 VKD0 12 VKD0 12B In the table before:

PL238 154B1

Table 20. The quality of spelled bread made with the addition of yeast fermented with fermented starter cultures

Sample name Loaf weight [g] Loaf volume [cm ³ ] Volume 100 g of bread [cm ³ ] Organoleptic assessment Crumb acidity [° kw] Average sum of points Quality class VSO 1 526 d ± 10 888 a ± 33 169 a ± 5 35.1 a ± 2.4 II 11 VSO 2 509 c ± 5 955 b ± 16 187b ± 4 35.5 a ± 1.8 II 9 VKDO 12 494 b ± 2 1082d ± 19 219 d ± 4 38.0 b ± 1.2 AND 4 VKDO 12B 492 b ± 3 1095 d ± 12 223 e ± 3 36.6 ab ± 2.9 AND 4

The table shows mean values ± standard deviations, a, b ... - mean values marked with different letters in the column differ statistically significantly at p <0.05

Spelled bread made of wholemeal flour with the addition of yeast produced on sourdough with the starter culture for the 12 and 12B set of microorganisms is characterized by a larger volume, significantly lower hardness and chewiness of the crumb compared to bread produced on the basis of spontaneous sourdoughs. The sourdough bread supplemented with the developed starter culture shows sufficient microbiological stability for this type of bread of 3 days in the case of Set 12 and 5 days for Set 12B.

The bread produced with the use of the developed starter culture obtained a significantly higher organoleptic evaluation compared to the bread based on spontaneous leaven, which was classified to the 2nd quality class.

Due to the obtained technological parameters, set 12B is especially recommended for spelled bread made of wholemeal flour with the participation of yeast.

PL238 154 B1

Table 21. Crumb texture profile on the day of baking spelled bread with the addition of yeast fermented with the participation of starter cultures

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The table shows mean values with standard deviations (SD), a, b ... - mean values marked with different letters in the column differ statistically significantly at p <0.05

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The first symptoms Mold growth was noted after 14 days of storage (2 cfu per loaf). Probably Penicillium sp. After 3 days of storage, mold growth (on the crumb and rind) of the genera Aspergillus, Penicillium and Mucor was noted. No changes indicating the development of amylolytic bacteria. No mold symptoms for 120 hours, on the 7th day of storage, both in thermostated samples and on whole loaves, a very strong growth of mold was noted No symptoms by 264 h (11 days) 360 h (15 days) 72 h (3 days) 120h (5 days) Sample name VSO 1 With OSA CM O > VKDO12B

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Table 25. Number of aerobic amylolytic bacteria (OLBA), aerobic spore amylolytic bacteria (OLBAP) and the number of mold and yeast (OLG) after 1, 5 and 7 days of storage of spelled bread produced with yeast fermented with starter cultures

Sample name OLBA OLBAP OLG 1 5 7 1 5 7 1 5 7 [CFU / g of bread] VSO1 0 0 0 0 0 0 0 0 0 VSO2 0 0 0 0 0 0 0 0 1.4x10 ² yeast 7KDO12 0 0 0 0 0 0 4.3 x10 ¹ 3.2x10 ³ 4.3x10 ⁴ VKDZ12B 0 0 0 0 0 0 7.5x10 ' 5.2x10 ³ 7x10 ⁵

Example 8

For the sourdoughs obtained on the wholemeal rye flour obtained in Example 3, microbiological analysis was carried out by culture-dependent methods by sowing 10-fold dilutions of the homogenized leaven on suitable substrates. In samples of leaven based on wholemeal flours, the number of microorganisms was determined, e.g.

total number of aerobic and anaerobic bacteria (on solid PCM with Aktidione, 72 h, at 30 ° C under aerobic and anaerobic conditions), thermophilic and mesophilic lactic acid bacteria (LAB) (on MRS medium, pH = 5.4; 72 h, at 42 ° C and 30 ° C in anaerobic conditions), yeast and mold (on YPG substrate, 5 days, at 25 ° C in aerobic conditions) and spore-forming bacteria (on LB substrate after sterilization of soup samples) , 48 h, at 30 ° C under aerobic conditions The results for the rye sourdoughs produced with the starter culture versus the commercial starter culture and the spontaneous sourdough after 72 h of fermentation are shown in Fig. 3.

Example 9

For the sourdoughs obtained on wholegrain spelled flour obtained in Example 4, microbiological analysis was performed by culture-dependent methods as described in Example 8. The results for sourdoughs from wholegrain spelled flour produced with the starter culture versus spontaneous sourdough after 72 h of fermentation are shown in Fig. 4.

Example 10

For the leaven obtained in Example 3 on wholemeal rye flour with the participation of Set 12 of starter culture and spontaneous rye leaven after 72 h of fermentation, the analysis of bacterial diversity was performed by methods independent of the culture. Metagenetic analysis of leaven samples was performed on the basis of bacterial metagenomic DNA obtained by enzymatic lysis with lysozyme and mutanolysin and extraction with phenol and chloroform. On the obtained DNA preparations, an amplification reaction was performed with the appropriate primers for the variable regions V3 and V4 of the genes encoding the 16S rRNA. The obtained amplicons were then sequenced by the NGS method (MiSeq Illumina platform). Sequencing readings were cleared and clustered using the MOTHUR program using the SILVA reference sequence database. The results of bioinformatics analysis show that the Firmicutes type (order of Lactobacillales) dominates in both sourdough samples, the share of which for Set 12 is 97%, and 98% for spontaneous sourdough. The taxonomic structure of bacteria in the studied leaven was presented at the level of the Lactobacillales order (Fig. 5). The most abundant species in both leaven is the genus Lactobacillus (80% and 87% of identified OTUs, respectively for Set 12 and spontaneous leaven).

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LITERATURE:

Bartnikowska, E. (2009). Contemporary views on the consumption of bread. NS. Piek. and Sugar. 1.4-11.

Ahn, H.-J., Kim, J.-H., Jo, C., Kim, M.-J., Byun, M.-W. (2004). Comparison of irradiated phytic acid and other antioxidants for antioxidant activity. Food Chem. 88, 173-178.

Chayen, J. (1994). The inositol phosphates: Chemical synthesis and biological significance. D. C. Billington. VCH: Weinheim, New York, Basel and Cambridge, xiv + 153 pages, 126DM (£ 52) (1993). Cell Biochem. Funct. 12, 77-78.

Chen, Q.C. and Li, B.W. (2003). Separation of phytic acid and other relatedinositol phosphates by high-performance ion chromatographyand its applications. J. Chromatogr. A 1018, 41-52.

De Angelis, M. (2003). Phytase activity in sourdough lactic acid bacteria: purification and characterization of a phytase from Lactobacillus sanfranciscensis CB1. Int. J. Food Microbiol. 87, 259-270.

Duliński, R., Cielecka, E.K., Pierzchalska, M., Żyła, K. (2015). Phytases improve myo-inositol bioaccessibility in rye bread: a study using an in vitro method of digestion and a caco-2 celi culture model. Food Technol. Biotechnol. 53, 66-72.

Gambuś, FI. and Litwinek, D. Bakery - why is it worth eating and what to choose?

http://dieta.mp. pl / rules / show.html? id = 74904.

Garcia-Estepa, R.M., Guerra-Hernandez, E., Garcia-Villanova, B. (1999). Phytic acid content in milled cereal products and breads. Food Res. Int. 32, 217-221.

Graf, E., and Eaton, J.W. (1990). Antioxidant functions of phytic acid. Free Radie. Biol. Med. 8, 61-69.

Johnson, J. and Linko, Y. (1964). Analysis of bread-flourcomponents. Oualitas Plantarum et Materia Vegatabilis 11.2 256.

Jurga, R. (2011). Promotion of flour and wholemeal bread in Poland. NS. Piek. and Sugar. 3, 10-11.

Kasim, A.B., and Edwards, FI.M. (1998). The analysis for inositol phosphate forms in feed ingredients. Sci Food Agric 76, 1-9.

Kawka, A. and Górecka, D. (2010). Comparison of the chemical composition of wheat-oat and wheat-barley bread with the participation of sourdoughs fermented with LV2 starter, FOOD. Science. Technology. Quality 3 (70), 44-55.

Lefebvre, D., Gabriel, V., Vayssier, Y., Fontagne-Faucher, C. (2002) Simultaneous FIPLC determination of sugars, organ acids and ethanol in sourdough process. Food Sci. Technol.

LEB 35, 407-414. Liu, B.-L., Rafiq, A., Tzeng, Y.-M., Rob, A. (1998). The induction and characterization of phytase and beyond. Enzyme Microb. Technol. 22, 415-424.

Park, H.-R., Ahn, H.-J., Kim, S.-H., Lee, C.-H., Byun, M.-W., Lee, G.-W. (2006). Determination of the phytic acid levels in infant foods using different analytical methods | DeepDyve. Food Control 17, 727-732.

Plaami, S. (1997). Myoinositol Phosphates: Analysis, Content in Foods and Effects in Nutrition. LWT - Food Sci. Technol. 30, 633-647.

Pontoppidan, K., Pettersson, D., Sandberg, A.-S. (2007). The type of thermal feed treatment influences the inositol phosphate composition. Anim. Feed Sci. Technol. 132, 137-147. Sandberg, A.-S., Carlsson, N.-G., Svanberg, U. (1989). Effects of Inositol Tri-, Tetra-, Penta-, and Hexaphosphates on In Vitro Estimation of Iron Availability. J. Food Sci. 54, 159-161.

Sandberg, A.-S., Brune, M., Carlsson, N.-G., Hallberg, L., Skoglund, E., Rossander-Hulthen, L. (1999). Inositol phosphates with different numbers of phosphate groups influence iron absorption in humans. Am. J. Clin. Nutr. 70, 240-246.

Wiseblatt, L. and Zoumut, H.F. (1963). Isolation, origin, and synthesis of a bread flavor constituent. Cereal Chem 40, 162-168.

Zhou, J.R., and Erdman, J.W. (1995). Phytic acid in health and disease. Crit. Rev. Food Sci. Nutr. 35,495-508.

PL238 154 Β1

She lived, Κ. (2005). Products of enzymatic conversion of phytates as functional food ingredients, in: Enzymatic modification of food ingredients. E. Kołakowski, W. Bednarski, S. Bielecki (ed.), AR in Szczecin: 495-510.

Sequence List:

<110> IBB <120> BACTERIAL STARTER CULTURE, LEADURE THIS CONTAINS, METHOD OF PRODUCTION OF BREAD AND APPLICATION OF BACTERIAL STARTER CULTURE OR ACID FOR PRODUCTION OF BREAD <130> <130> <211> 16 <212> DNA <213> artificial <220>

<2 23> RAPD-GACA primer <400> 1 gacagacaga cagaca 16 <210> 2 <211> 10 <212> DNA <213> artificial <220>

<223> RAPD-B10 starter <400> 2 ctgctgggac 10

Claims (17)

Patent claims 1. Bacterial starter culture containing lactic acid bacteria in the form of biomass, characterized in that the composition contains the Lactobacillus plantarum strain deposited as B / 00117, Lactobacillus plantarum deposited as B / 00118 and Lactobacillus brevis deposited as B / 00119 and Weisella confusa deposited as / 00120. 2. The bacterial starter culture according to claim 1, The method of claim 1, wherein the ratio of Lactobacillus plantarum strains deposited as B / 00117 to Lactobacillus plantarum deposited as B / 00118 to Lactobacillus brevis deposited as B / 00119 to Weisella confus deposited as B / 00120 is 1: 1: 1: 1, respectively. 3. A bacterial starter culture according to any one of claims 1 to 5. 1-2, characterized in that it contains at least 1 × 10 ¹⁰ cfu / ml of bacteria in the biomass. 4. A sourdough made from wholemeal or whole grain flour, characterized in that it comprises the starter culture according to any one of claims 1 to 3; 1-3. 5. The sourdough according to claim 1 A method according to claim 4, characterized in that the leaven is made of wholemeal rye flour and is intended for the production of bread without the addition of yeast. 6. The sourdough according to claim 1 4. A method according to claim 4, characterized in that the leaven is made of wholemeal rye flour and is intended for the production of bread with yeast addition. 7. The sourdough according to claim 1 6. A method according to claim 6, characterized in that the starter culture consists essentially of exclusively Lactobacillus plantarum strains deposited as B / 00117 and Lactobacillus plantarum PL 238 154 B1 deposited as B / 00118 and Lactobacillus brevis deposited as B / 00119 and Weisella confusa deposited as B / 0010 in a ratio of 1: 1: 1: 1. 8. The sourdough according to claim 1 4. A method according to claim 4, characterized in that the leaven is made of spelled flour. 9. Process for the production of bread, characterized in that it comprises the step of adding a starter culture according to any one of claims 1 to 9. 1-3 or of the sourdough according to any one of claims 1 to 3 4-8. 10. Use of a starter culture according to any one of claims 1 to 10. 1-3 or of the sourdough according to any one of claims 1 to 3 4-8 for making bread. 11. New Lactobacillus plantarum bacterial strain deposited in PCM under No. B / 00117. 12. New Lactobacillus plantarum bacterial strain deposited in PCM under No. B / 00118. 13. New Lactobacillus brevis bacterial strain deposited in PCM under No. B / 00119. 14. New Weisell confus bacterial strain deposited in PCM under number B / 00119. 15. A bacterial composition comprising at least one bacterial strain according to claims 11-14. A method of producing bread, characterized in that it comprises the step of adding a new bacterial strain according to claim 1. 11-14 and / or the bacterial composition according to claims 11-14 and / or 15. 17. The use of a new bacterial strain according to claim 1 1-14 and / or a bacterial composition according to claims 1-14. 15 for making bread.