A method for obtaining an ingredient and flour formulation enriching the protein content in bakery products and the ingredient / flour formulation obtained by the method

Abstract

The present invention relates to a method (100) for obtaining an ingredient and flour formulation enriching the protein content in bakery products, by making use of pomegranate peel and pomegranate seed pomace that have negative effects on the environment and low added value, and to a bakery product rich in protein content obtained by the method (100).

Description

[0001] DESCRIPTION

[0002] A METHOD FOR OBTAINING AN INGREDIENT AND FLOUR FORMULATION ENRICHING THE PROTEIN CONTENT IN BAKERY PRODUCTS AND THE INGREDIENT / FLOUR FORMULATION OBTAINED BY THE METHOD

[0003] Technical Field

[0004] The present invention relates to a method for obtaining an ingredient and flour formulation enriching the protein content in bakery products, by making use of pomegranate peel and pomegranate seed pomace that have negative effects on the environment and low added value, and to a bakery product rich in protein content obtained by the method.

[0005] Background of the Invention

[0006] With population growth, food demand is increasing, and consequently, production in the food industry must be increased. This increase in production also increases the need for land, raw materials, energy, water, and other resources. In addition, as noted in the report by the Intergovernmental Panel on Climate Change (IPCC), 21-37% of greenhouse gas emissions, a critical issue in regard to global climate change, originate from the food industry. Today, proteins, which have an important place in the food industry and are essential for humans to maintain their vitality, are conventionally obtained from plants and animals. Due to the fact that these resources are limited and that raw material costs, as well as water, land, and energy demands are high, they are neither environmentally sustainable nor accessible to all socioeconomic groups, creating the need for a new alternative protein source. In this context, there is a need to develop a production technology for an alternative protein source in the food industry that is sustainable and has minimal raw material and resource consumption.In Turkey, the fruit juice industry is among the sectors that generate the most waste during production. The waste obtained during production (fruit pomace, peels, etc.) is mostly left in open areas, incinerated, or used in animal feed or compost production. This type of practice poses a threat to the environment and human health and can lead to the generation of secondary waste. Fruit juice industry waste is a rich source of carbon and nitrogen for microorganisms; this presents a significant potential for making use of this waste in fermentation processes. It is possible both to develop products with high added value and to produce proteinrich, functional foods from these waste materials. In this regard, proteins produced through fermentation using microorganisms and extracted from the medium have the potential to be included as ingredients in food formulations.

[0007] Turkey is one of the leading countries in the world in pomegranate production, and the processing of pomegranates in the industry generates significant amounts of waste, such as peels and seeds. This waste has the potential to provide both economic and environmental benefits by being sustainably reused by means of fermentation technologies. For this reason, there is a need to produce an ingredient from pomegranate peel and seed pomace to be used in the food industry, and even to produce ingredients having high protein content that can be used in the baking industry with the aim of increasing their nutritional value.

[0008] The Chinese patent document no. CN113502233, an application included in the state of the art, discloses a method for extracting soluble dietary fibers from pomegranate peel residues and seeds. The method comprises the steps of drying and crushing the pomegranate peel residues and the seed mixture to prepare pomegranate peel residues and seed powder; mixing the pomegranate peel residue and seed powder with deionized water to prepare pomegranate peel residue and seed feed liquid, adding glucose and the like into the pomegranate peel residue and seed feed liquid, inoculating lactobacillus casei and lactobacillus acidophilus for culturing, thereby obtaining pomegranate peel residue and seed fermentation liquid;repeatedly freezing and thawing the pomegranate peel residue and seed fermentation liquid, restoring to normal temperature and normal pressure, and sterilizing; centrifuging and suction-filtering the sterilized pomegranate peel residue and seed fermentation liquid to obtain a first filter residue and first filtrate, performing enzymolysis on the first filter residues for multiple times, centrifuging and suction-filtering to obtain second filtrate, combining the first filtrate and the second filtrate, performing vacuum rotary evaporation and concentration to obtain concentrated liquor, adding absolute ethyl alcohol into the concentrated liquor, standing overnight, centrifuging, reserving precipitate, and drying and crushing to obtain the soluble dietary fibers in the pomegranate peel residues and seeds. The inventive method can effectively extract the soluble dietary fibers in the pomegranate peel residues and seeds.

[0009] Summary of the Invention

[0010] An object of the present invention is to realize a method for obtaining an ingredient and flour formulation enriching the protein content in bakery products, by making use of pomegranate peel and pomegranate seed pomace that have negative effects on the environment and low added value, and a bakery product rich in protein content obtained by the method.

[0011] Another object of the present invention is to realize a production method for obtaining an ingredient rich in protein content from pomegranate peel and seed pomace by applying fermentation technology and bioprocess design.

[0012] A further object of the present invention is to realize a method for obtaining a product usable as food for human consumption without applying any chemical treatment or adding any chemicals to pomegranate peel and pomegranate seed pomace during production.A further object of the present invention is to realize a method for reducing dependence on imported commercial strains and enabling more effective use of local resources by using a local and indigenous Saccharomyces cerevisiae strain when obtaining a bakery product rich in protein content from pomegranate peel and seed pomace.

[0013] A further object of the present invention is to realize a flour formulation to be used in bakery products with the produced ingredient rich in protein content.

[0014] Detailed Description of the Invention

[0015] “A Method for Obtaining an Ingredient and Flour Formulation Enriching the Protein Content in Bakery Products and the Ingredient / Flour Formulation Obtained by the Method” realized to fulfil the objectives of the present invention is shown in the figures attached, in which:

[0016] Figure l is a flowchart of the inventive method.

[0017] Figure 2 is the pH, cell count (CFU / mL), amount of protein produced (% w / w), biomass yield (mg / mL), and changes in oxygen saturation (pO2) analyzed in the fermentation carried out by using S. cerevisiae strain coded HUF16M3G11088 from pomegranate seed-pomegranate peel (50-50% w / w) pomace.

[0018] 100. Method

[0019] 101. Preparing a medium with pomegranate peel and pomegranate seed pomace

[0020] 102. Carrying out fermentation with pomegranate peel and pomegranate seed pomace in the prepared medium

[0021] 103. Obtaining an ingredient rich in protein content by carrying out biomass purification after fermentation

[0022] 104. Preparing flour formulation to be used in bakery products with the protein-containing ingredientThe inventive method (100) for obtaining an ingredient and flour formulation enriching the protein content in bakery products, by making use of pomegranate peel and pomegranate seed pomace that have negative effects on the environment and low added value comprises the steps of

[0023] preparing a medium with pomegranate peel and pomegranate seed pomace (ioi);

[0024] carrying out fermentation with pomegranate peel and pomegranate seed pomace in the prepared medium (102);

[0025] obtaining an ingredient rich in protein content by carrying out biomass purification after fermentation (103); and

[0026] preparing flour formulation to be used in bakery products with the proteincontaining ingredient (104).

[0027] At the step of preparing a medium with pomegranate peel and pomegranate seed pomace (101) of the inventive method (100), pomegranate peel and pomegranate seed pomace are weighed at a ratio of 45-55% (w / w), and pure water is added at a ratio of 1 / 3 to 1 / 5 (g pomace / mL pure water); then, mechanical disintegration process is applied for 3-5 minutes; coarse filtration process is applied to the resulting solution by passing it through filter with a diameter of 1.5 mm; sterilization process is applied to the resulting 500-1000 mL filtrate at 121°C for 15 minutes; and as a result of the sterilization process, the medium / cell solution to be used in fermentation is obtained.

[0028] At the step of carrying out fermentation with pomegranate peel and pomegranate seed pomace in the prepared medium (102) of the inventive method (100), inoculation is performed at a cell concentration of approximately 1011CFU / mL by introducing a cell solution prepared with the Saccharomyces cerevisiae strain with the local HUF 16M3G11088 code into a fermenter having at least one Rushton-type mixer and temperature, pH, dissolved oxygen (pCh), and foam probes; batch fermentation is performed such that the fermentation parameters are 72 hours totalfermentation duration, 28°C temperature, 400 rpm constant stirring speed, and 0,5 1 / min air supply; dissolved oxygen in the medium is fixed at 30% saturation by being regulated by the air flow and stirring speed.

[0029] During fermentation, the cell count is observed to increase rapidly over a period of 0-10 hours, reaching levels of approximately 7,3 log (CFU / mL) (Figure 2). Between 20 and 72 hours, the cell concentration remains constant. A decrease in pH values is observed in direct proportion to cell growth; as a result of fermentation, the pH drops to 3.50. The protein concentration in the medium (w / w %) increases between 10 and 40 hours, reaching an average concentration of 16% (w / w). As a result of fermentation, protein production continues to increase, reaching an average of 18% (w / w). Biomass yield (mg / mL) increases after 20 hours, reaching an average of 25 mg / mL. A change in dissolved oxygen saturation (pCL) is also observed in direct proportion to cell growth.

[0030] At the step of obtaining an ingredient rich in protein content by carrying out biomass purification after fermentation (103) of the inventive method (100), the solution obtained as a result of fermentation is centrifuged at 6.000 rpm for 5 minutes and washed twice with sterile pure water, thereby obtaining wet biomass from the samples; then, drying process is applied to the resulting wet biomasses for 24 hours using a lyophilizer, and after the drying process, the protein ingredient is obtained in the form of lyophilized dry powder.

[0031] Nutritional analysis (dry matter and moisture, and protein), safety analysis (amount of nucleic acids), primary metabolite analysis, and a life cycle assessment (LCA) were performed on the protein ingredient in the form of a dry powder obtained as a result of the lyophilization process.

[0032] The methods used for the analyses are given below. The results of the dry matter and moisture, protein, and nucleic acid analyses are shown in Table 1.Nutritional and Safety Analyses

[0033] For the nutritional analysis of the samples, dry matter and moisture analysis and protein analysis were performed. For safety analysis, the nucleic acid (DNA) amount analysis was performed on the samples.

[0034] In dry matter and moisture analysis, a moisture analyzer with an infrared lamp heating system was used to determine the moisture% content. Approximately 0,5 grams of sample was weighed into tared aluminum containers. The device was set to 105°C. When the moisture% value stabilized on the device screen, the drying process was terminated and the result was recorded.

[0035] In order to determine the protein content in the samples, protein extraction was first performed on the samples. For protein extraction, 0,5 g of sample was weighed and placed into 15 mL Falcon tubes. A 0,1 M NaOH solution was added to the sample in 30 mL of 3,5% NaCl. Then, extraction process was applied to the samples with an ultrasonic homogenizer for 6 minutes. The resulting extract was incubated in a water bath at 60°C for 90 minutes. Then, centrifuge process was applied at 4000 xg and 4°C for 30 minutes. The supernatant obtained from the centrifuge was stored at -20°C until the protein content determination. The BCA Protein Assay method was used in protein analysis. From the supernatants obtained as a result of protein extraction from the samples, 25 pL was placed in 96-well sterile cell culture petri dishes, and 200 pL of BCA reagent was added onto them. The samples were then incubated at 37°C for 30 minutes. As a result of the incubation, absorbance measurements were taken at 562 nm in a microplate reader. The obtained absorbance values were plotted on a calibration curve, and the protein content in the samples was calculated in mg / mL.

[0036] The amount of DNA in the sample was analyzed in order to determine the total nucleic acid amount. For this purpose, DNA isolation was performed by using GeneMATRIX Bacterial & Yeast Genomic DNA Purification Kit of EURX brand.In order to lyse the DNA, first, Lyse BG buffer was added to 10 mg of powder sample in 300 pL of 0,001% (v / v) P-mercaptoethanol. The solution was centrifuged at 11.000 xg for 1 minute. After centrifugation, Lyse BG buffer was added again in 0,001% (v / v) P-mercaptoethanol, and mixing with a pipette was performed. Then, 30 pL of Lyticase (20 mg / mL) and 2 pL of RNase A were added. The tubes were incubated at 30°C for 30 minutes by using a dry incubator. After incubation, 15 pL of Proteinase K solution was added to the tubes for the isolation step, and vortexed for 3 seconds. Then, the samples were incubated in a dry incubator at 55°C for 30 minutes. During the incubation period, the tubes were shaken every 10 minutes. Then, 350 pL of SOL BG was added to the samples and vortexed for 3 seconds. The samples were incubated at 55°C for 5 minutes. As a result of the incubation, the samples were vortexed for 15 seconds and centrifuged at 11.000 xg for 2 minutes. The resulting supernatant was then transferred to a DNA binding column. The columns containing the supernatant were centrifuged at 11.000 xg for 1 minute. After centrifugation, 450 pL of Wash BGX buffer was added, and the centrifugation process was repeated at 11.000 xg for 1 minute. This process was repeated a total of two times. Finally, the column containing the supernatant was centrifuged at 11.000 xg for 1 minute without adding any chemicals. The columns were then placed into a new microcentrifuge tube, and 50 pL of Elution buffer was added. After the addition, the tubes were kept at room temperature for 2 minutes. As a final step, the supernatant obtained after centrifugation performed at 11.000 xg for 1 minute was used in the determination of DNA content.

[0037] The analysis results for moisture%, protein, and nucleic acid content of the lyophilized protein ingredient in the form of dry powder, obtained as a result of fermentation carried out by using the S. cerevisiae strain with the code HUF16M3G11088 from pomegranate seed-peel (50-50% w / w) waste, are shown in Table 1.

[0038] Table 1. Moisture (%), protein (%), and nucleic acid (%) content of the lyophilized protein ingredient in the form of dry powder, obtained as a result of fermentationcarried out by using the S. cerevisiae strain with the code HUF16M3G11088 from pomegranate seed-peel (50-50% w / w) waste.

[0039] Humidity (%) Protein (%) Nucleic Acid (%)

[0040] 7,00±1.27 18,47 ±0,65 0.20±0.00

[0041]

[0042] Primary Metabolite Analysis

[0043] Gas Chromatography and Mass Spectrometry (GC-MS) were used with the aim of detecting primary metabolites in the produced microbial protein in the form of powder. For the analysis, 800 pL of 100% methanol, 100 pL of the internal standard ribitol (1 mg / mL), and 100 pL of water for chromatography were added onto 100 mg of the sample in 1,5 mL. The tube was then vortexed for 1 minute. In order to perform the extraction, the samples were treated in an ultrasonic bath at 35°C for 60 minutes. During this process, vortexing was performed every 10 minutes. The resulting mixture was centrifuged at 10.000 g for 15 minutes at 4°C. The supernatant obtained after centrifugation was placed in a new microcentrifuge tube.

[0044] 800 pL of 100% methanol and 200 pL of water for chromatography were added to the remaining pellet. The tube was then vortexed again for 1 minute. The tubes were treated again in an ultrasonic bath and centrifuged. The supernatant was mixed with the supernatant obtained from the previous step for 1 minute with the help of a vortex. The mixture was centrifuged at 10.000 xg at 4°C for 15 minutes. After centrifugation, 1 mL of the liquid supernatant was placed in a 1,5 mL amber-colored GC vial and dried under a nitrogen gas stream. The dried sample was dissolved in methoxylamine hydrochloride (20 mg / mL) dissolved in 200 pL of pyridine for the derivatization process. Then, the samples were shaken at 400 rpm for 90 minutes at 37°C. After adding 200 pl of N-methyl-N-(trimethylsilyl)trifluoroacetamide onto the sample at the end of the shaking step, the incubation was carried out by shaking again at 400 rpm for 90 minutes at 37°C. The resulting solution was stored at -20°C before the analysis.The analysis was performed in an Agilent 7890B GC / 5977A Series MSD Systems device. An Agilent 122-5532G DB-5MS+DG 30 m * 250 pm x 0.25 pm column was used in the analysis. The injection volume was set to 0.5 pl and the split ratio to 50:1. Samples were injected into the system at a flow rate of 1 mL / min with helium as the carrier gas. The solvent delay was 2 minutes; the oven temperature was initially set to 80°C for 1 minute and then programmed to remain at 290°C for 7 minutes after reaching 290°C at a heating rate of 5°C / min. The mass range was set between 10-600 (amu).

[0045] As a result of the analysis, amino acids, fatty acids, organic acids, sugars, phenolic compounds, vitamins, and components belonging to the phytosterol group were found in the sample (Table 2).

[0046] Table 2. Primary metabolites found in the lyophilized protein ingredient in the form of dry powder, obtained as a result of fermentation carried out by using the S. cerevisiae strain with the code HUF 16M3G11088 from pomegranate seed-peel (50-50% w / w) waste.

[0047] No Amino Acids and Derivatives Retention Durations (Minutes)

[0048] 1 L-Alanine, 2TMS derivative 6.719

[0049] 2 L-Threonine, 3TMS derivative 13.404

[0050] 3 L-5 -Oxoproline, 2TMS 16.601

[0051] No Fatty Acids and Derivatives

[0052] 1 Myristic acid, TMS derivative 23.989

[0053] 2 Undecanedioic acid, 2TMS derivative 24.176

[0054] 3 Palmitelaidic acid, TMS 27.460

[0055] 4 Palmitic Acid, TMS derivative 27.874

[0056] 5 9,12-Octadecadienoic acid (Z,Z)-, 30.848

[0057] TMS derivative

[0058] 6 Oleic Acid, (Z)-, TMS derivative 30.961

[0059] 7 Stearic acid, TMS derivative 31.449

[0060] Glycerolipids

[0061]

[0062] 1 1 -Monopalmitin, 2TMS derivative 37.035 2 Glycerol monostearate, 2TMS 39.875 derivative

[0063] No Organic Acids and Derivatives

[0064] 1 Propanoic acid, 2-(methoxyimino)-, 5.683 trimethyl silyl ester

[0065] 2 Lactic Acid, 2TMS derivative 5.880 3 Butanedioic acid, 2TMS derivative 11.643 4 Glyceric acid, 3TMS derivative 12.038 5 Malic acid, 3TMS derivative 15.947 6 2-Isopropylmalic acid, 0- 17.918 trimethylsilyl-, bis(trimethylsilyl)

[0066] ester

[0067] 7 Pentanedioic acid, 2- 18.032 [(trimethylsilyl)oxy]-,

[0068] bi s(trimethyl silyl) ester

[0069] 8 4-Hydroxybenzoic acid, 2TMS 19.138 derivative

[0070] 9 Citric acid, 4TMS derivative 23.384 No Sugars and Derivatives

[0071] 1 Erythritol, 4TMS derivative 16.454 2 2,3,4-Trihydroxybutyric acid 17.684 tetrakis(trimethylsilyl) deriv., (, (R*,

[0072] R*)-)

[0073] 3 D-Arabinose, tetrakis(trimethylsilyl) 20.023 ether, ethyloxime (isomer 2)

[0074] 4 Fructose benzoyl oxime, 20.350 pentakis(trimethyl silyl)- 5 Xylitol, 5TMS 21.286 6 D-(+)-Ribono-l,4-lactone (R, S, R)-, 21.519 3TMS derivative

[0075] 7 Ribonic acid, 2,3,4,5-tetrakis-O- 22.214 (trimethyl silyl)-, trimethyl silyl ester

[0076] 8 D-(-)-Ribofuranose, 22.321 tetrakis(trimethylsilyl) ether (isomer

[0077] 2)

[0078] 9 D-Psicose, pentakis(trimethylsilyl) 24.460 ether, methyloxime (syn)

[0079] 10 D-(+)-Gluconolactone, 4TMS 24.687 11 d-Galactose, 2,3,4,5,6-pentakis-O- 24.814

[0080] (trimethyl silyl)-, o-methyloxyme,

[0081] (IE)- 12 Galactonic acid, y-lactone, 4TMS 24.901

[0082]

[0083] 13 d-Galactose, 2,3,4,5,6-pentakis-O- 24.951

[0084] (trimethyl silyl)-, o-methyloxyme,

[0085] (1Z)- 14 Sedoheptulose, 6TMS derivative 25.486

[0086] 15 D-Mannitol, 6TMS 25.596

[0087] 16 D-Gluconic acid, 6TMS 26.952

[0088] 17 Myo-Inositol, 6TMS 28.622

[0089] 18 D-Myo-Inositol, 1,2,4,5,6-pentakis- 34.229

[0090] O-(trimethylsilyl)-, bi s(trimethyl silyl)

[0091] phosphate

[0092] 19 D-Trehalose 8TMS 39.300

[0093] 20 D-(+)-Cellobiose, 41.108 octakis(trimethyl silyl) ether,

[0094] methyloxime (isomer 2)

[0095] No Alcohols and Derivatives

[0096] 1 2,3-Butanediol, 2TMS derivative 5.406

[0097] 2 Ethanolamine, 3TMS derivative 10.434

[0098] 3 Glycerol, 3TMS derivative 10.654

[0099] No Phenolic Compounds

[0100] 1 Gallic acid, 4TMS derivative 26.150

[0101] 2 Epigallocatechin (6TMS) 41.837

[0102] No Vitamins

[0103] 1 y-Tocopherol, TMS derivative 42.745

[0104] No Phytosterols

[0105] 1 P-Sitosterol, TMS derivative 48.254

[0106]

[0107] Life Cycle Assessment (LCA)

[0108] The life cycle assessment (LCA) of the lyophilized protein ingredient in the form of dry powder was carried out based on the IPPC’s report titled “AR6 Climate Change 2021: The Physical Science Basis” and with the SimaPro 9.4 software. In production, the carbon footprint (CO2 emission amount) of the product was determined by calculating emission factors for all inputs / outputs during the production process by using the SimaPro 9.4 software.

[0109] When all the data was entered into the system, 0,00354 kg CO2 eq. / gr of product for the raw material preparation step; 0,00198 kg CO2 eq. / gr of product for theproduction of lyophilized microbial protein in the form of dry powder with fermentation; and a product emission rate of 0,105 kg CO2 eq. / gr for the produced microbial protein were calculated. It was determined that the emission amount of the final product is 0, 11 kg CO2 eq. / gr product.

[0110] At the step of preparing flour formulation to be used in bakery products with the protein-containing ingredient (104) of the inventive method (100), a lyophilized protein ingredient in the form of a powder, produced from pomegranate peel and pomegranate seed pomace, is added at a rate of 30-35% (w / w) to a mixture consisting of wheat flour, sugar, starch, cocoa powder, sodium bicarbonate, skim milk powder, and vanillin and a flour formulation is obtained.

[0111] Nutritional (dry matter and moisture, protein), safety (amount of nucleic acid), and color analyses were performed on the flour formulation created to be used in bakery products.

[0112] Nutritional and Safety Analyses

[0113] The methods mentioned above under the title of Nutritional and Safety Analyses were used to determine the moisture% content and nucleic acid amount of the product. The Dumas protein analysis method was applied to determine the protein content. When calculating the protein content of the product, the nitrogen factor (N) was taken as 6.25. The nutritional and safety results for the protein ingredient are shown in Table 3.

[0114] Table 3. Moisture (%), protein (%), and nucleic acid (%) content of the flour formulation created by using protein ingredients.

[0115] Humidity (%) Protein (%) Nucleic Acid (%) 6.25 ± 0.05 9,75 ±0,34 0.061± 0.00

[0116]

[0117] Color Analysis

[0118] A color analysis was performed in accordance with the Hunter L*a*b system in order to demonstrate the color difference of the product compared to the control group. The L* value represents the degree of lightness (100: white, 0: black), the a value represents the redness-greenness (60+ : red, 60-: green), and the b value represents the yellowness-blueness (60+: yellow, 60-: blue). The AE value represents the proximity or distance from the color of the control group used for comparison. The following formula was used to calculate the AE value.

[0119]

[0120] AF = V(AL)2+ (A®)2A (M)2

[0121] AL: The difference between the L* values of the control and the sample

[0122] Aa: The difference between the a* values of the control and the sample

[0123] Ab: The difference between the b* values of the control and the sample

[0124] In this context, the results of the color analyses of the protein ingredient are shown in Table 4. Based on the results obtained, it was detected that the created flour formulation has light (L: 70.4+0.75), slightly reddish (a: 3.2+0.19), and yellowish (b: 5.9+0.34) tones, and that the color is distributed homogeneously (E: 2.1+0.56).

[0125] Table 4. Color analysis results for the flour formulation created by using the protein ingredient.

[0126] Color Analysis

[0127] Average L* Average a* Average b* Average AE

[0128]

[0129] 70.4+0.75 3.2+0.19 5.9+0.34 2.1+0.56

[0130] Furthermore, the said invention relates to an ingredient containing a high protein content, obtained by following the steps of the aforementioned method (100), and a flour formulation to be used as a bakery product prepared with this ingredient.Within these basic concepts; it is possible to develop various embodiments of the inventive “A Method for Obtaining an Ingredient and Flour Formulation Enriching the Protein Content in Bakery Products and the Ingredient / Flour Formulation Obtained by the Method”; the invention cannot be limited to examples disclosed herein and it is essentially according to claims.

Claims

CLAIMS1. A method (100) for obtaining an ingredient and flour formulation enriching the protein content in bakery products, by making use of pomegranate peel and pomegranate seed pomace that have negative effects on the environment and low added value; characterized by the steps ofpreparing a medium with pomegranate peel and pomegranate seed pomace (ioi);carrying out fermentation with pomegranate peel and pomegranate seed pomace in the prepared medium (102);obtaining an ingredient rich in protein content by carrying out biomass purification after fermentation (103); andpreparing flour formulation to be used in bakery products with the proteincontaining ingredient (104).

2. A method (100) according to Claim 1; characterized in that at the step of preparing a medium with pomegranate peel and pomegranate seed pomace (101), pomegranate peel and pomegranate seed pomace are weighed at a ratio of 45-55% (w / w), and pure water is added at a ratio of 1 / 3 to 1 / 5; then, mechanical disintegration process is applied for 3-5 minutes; coarse filtration process is applied to the resulting solution by passing it through filter with a diameter of 1.5 mm; sterilization process is applied to the resulting 500-1000 mL filtrate at 121°C for 15 minutes; and as a result of the sterilization process, the medium / cell solution to be used in fermentation is obtained.

3. A method (100) according to Claim 1 or 2; characterized in that at the step of carrying out fermentation with pomegranate peel and pomegranate seed pomace in the prepared medium (102), inoculation is performed at a cell concentration of approximately 1011CFU / mL by introducing a cell solution prepared with the Saccharomyces cerevisiae strain with the local HUF16M3G11088 code into a fermenter having at least one Rushton-type mixer and temperature, pH, dissolvedoxygen (pCh), and foam probes; batch fermentation is performed such that the fermentation parameters are 72 hours total fermentation duration, 28°C temperature, 400 rpm constant stirring speed, and 0,5 1 / min air supply; dissolved oxygen in the medium is fixed at 30% saturation by being regulated by the air flow and stirring speed.

4. A method (100) according to any one of the preceding claims; characterized in that at the step of obtaining an ingredient rich in protein content by carrying out biomass purification after fermentation (103), the solution obtained as a result of fermentation is centrifuged at 6.000 rpm for 5 minutes and washed twice with sterile pure water, thereby obtaining wet biomass from the samples; then, drying process is applied to the resulting wet biomasses for 24 hours using a lyophilizer, and after the drying process, the protein ingredient is obtained in the form of lyophilized dry powder.

5. A method (100) according to any one of the preceding claims; characterized in that at the step of preparing flour formulation to be used in bakery products with the protein-containing ingredient (104), a lyophilized protein ingredient in the form of a powder, produced from pomegranate peel and pomegranate seed pomace, is added at a rate of 30-35% (w / w) to a mixture consisting of wheat flour, sugar, starch, cocoa powder, sodium bicarbonate, skim milk powder, and vanillin and a flour formulation is obtained.

6. An ingredient containing the protein content obtained by following the steps of the above method (100), and a flour formulation to be used as a bakery product prepared with this ingredient.

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