Study regarding the improvment of quality and nutritional parameters in aglutenic functional products obtained through lactic sourdough fermentation

Transcription

1 PhD THESIS SUMMARY Study regarding the improvment of quality and nutritional parameters in aglutenic functional products obtained through lactic sourdough fermentation PhD student Maria Simona Chiș Scientific coordinator Prof.univ. dr. Sevastița Muste CLUJ-NAPOCA, 2017

2 Study regarding the improvment of quality and nutritional parameters in aglutenic functional products obtained through lactic sourdough fermentation INTRODUCTION New consumers demand for products with improved nutritional qualities and health benefits, which involves new challenges for the bakery industry. Moreover, wheat or rye bread products are problematic due to the growth number of celiac disease sufferers. Therefore there is a new market for products made from grain alternatives such as rice, corn, sorghum, millet, or pseudo-cereals such as buckwheat (Fagopyrum esculentum Mönch), amaranth (Amaranthus caudatus L.) and quinoa (Chenopodium quinoa Willd), possibly roots containing starch, as well as manioc (Manihot esculenta Crantz). Another possibility to improve the nutritional quality of aglutenic and functional products is the utilization of sourdough. Sourdough is a mixture of flour and water fermented with lactic acid bacteria and /or yeast. The term lactic acid bacteria (LAB) describes a group of gram positive bacteria with metabolic and physiological characteristics. LAB are optional aerobic or anaerobic organisms, of the genus Lactobacillus, which produce metabolic energy from fermentable carbohydrates by phosphorylation at the substrate level. Lactobacillus plantarum is a facultative heterofermentative strain, mostly associated with plant matrices, therefore present in many types of food fermentations (eg cocoa beans, cabbage). Its wide use is supported by increasing acidification speed and its good adaptation due to the high flexibility metabolism in challenging environments with other lactic bacteria and / or yeast strains. Lactobacillus spicheri, is a heterofermentative strain, wich belong to the Lactobacillus Buchneri group, used in sourdough fermentation. The strain used in this study, Lactobacillus spicheri DSM (Leibniz Institute-German Collection of Microorganisms and Cell Cultures) 15429, was discovered by MEROTH et al., 2004 and isolated from industrially processed rice sourdough. It bears the name of the german researcher Gottfried Spicher who contributed to the chemical, microbial and technological characterization of the wheat and rye sourdoughs, opening new research horizons. The research in this paper was carried out in Food Quality Control Laboratories, the LICSA Laboratory, Bakery Pilot Station at the Faculty of Food Science and Technology, the Molecular Nutrition Laboratory (Genomics and Proteomics), the Beekeeping Biotechnology Laboratory within the Institute of Life Sciences, USAMV Cluj-Napoca. II

3 Maria Simona Chiș AIM AND OBJECTIVES AIM OF THE THESIS The research for the paper,, Study regarding the improvment of quality and nutritional parameters in aglutenic functional products obtained through lactic sourdough fermentation have as main purpose the elaboration of a new functional aglutenic product by using lactic acid bacteria and studying the influence of lactic acid bacteria on the nutritional quality value of the sourdough as well as on the final product. OBJECTIVES In order to accomplish the main purpose, were addressed three major research directions, etablished as the objectives of this thesis: quantification of the main chemical compounds of rice, quinoa and buckwheat flours, oatmeal. studying the qualitative and nutritional influence of lactic acid bacteria on quinoa and rice sourdoughs during fermentation obtaining an innovative functional product for people with special needs STRUCTURE OF THE THESIS Thesis entitled,,study regarding the improvment of quality and nutritional parameters in aglutenic functional products obtained through lactic sourdough fermentation is divided in two main parts, the first part,,,the current state of knowledge regarding the sourdough, the aglutenic functional products, and the chemical composition of raw materials used in manufacturing of the final products ", including the literature study, and the second part,,,original contributions to knowledge development in the field of sourdough and aglutenic products ", including personal contributions, results, conclusions, elements of originality and research perspectives. The total number of tables in this thesis is 25 and the total number of figures is 66. The first part,,,the current state of knowledge regarding the sourdough, the aglutenic functional products, and the chemical composition of raw materials used in manufacturing the final products ", is structured in two chapters. Chapter 1: LACTIC SOURDOUGH comprises four subchapters presenting the results obtained after an extensive literature study, namely: generalities regarding lactic acid bacteria, their importance in food industry, their role in the sourdough and implicitly in the final product, celiac disease and the consequences of an aglutenic diet, evidencing the need for the development of functional gluten products. III

4 Study regarding the improvment of quality and nutritional parameters in aglutenic functional products obtained through lactic sourdough fermentation Chapter II: PRESENTATION AND CHARACTERIZATION OF RAW MATERIALS, which contains 8 subchapters presenting the chemical composition of raw materials used in this thesis. Quinoa flour is a functional raw material due to the perfect content of amino acids and protein, as well as other bioactive compounds with a functional role: folic acid, minerals, phenols, phytosterols, vitamins. Also, rice flour is a valuable raw material due to its protein content (with a high degree of digestibility of 93%), vitamins and minerals. Part two, PERSONAL CONTRIBUTION, is structured in four chapters. Chapter III AIM AND ABJECTIVES, includes the specific purpose and general objectives of the thesis. Chapter IV MATERIALS AND METHODS focuses on presenting the experimental material and three working protocols related to research in the 10 sub-chapters of it. CHAPTER V, RESULTS AND DISCUSSIONS disseminates in three subchapters the results obtained regarding the analysis of the raw materials used, the analysis of the sourdough, the analysis of the final products as well as the statistical interpretation of the obtained data. Chapter III. MATERIALS AND METHODS 3.1. BIOLOGICAL MATERIAL PERSONAL CONTRIBUTIONS The biological material used to perform the experiments consists of samples of rice flour, quinoa seed, buckwheat flour, inulin, oatmeal, potato starch, egg, maple syrup, coconut oil, lactic acid bacteria: Lactobacillus plantarum ATCC (American Type Culture Collection) 8014 and the Lactobacilus spicheri DSM (Leibniz Institute-German Collection of Microorganism and Cell Cultures )15429 and the finished product resulting from the processing of raw materials - Figure 1. Raw materials such as quinoa seeds, whole rice flour, buckwheat flour, oatmeal, potato starch, eggs, maple syrup and coconut were purchased from specialized stores from Cluj-Napoca. IV

5 Maria Simona Chiș Fig.1. Experimental material aglutenic muffins 3.2. METHODS In order to achieve the proposed objectives, were modeled three experimental protocols, structured according to three major research directions Quantification of the main chemical compounds of the raw materials used in manufacturing the final product In this sense, the following specific objectives have been developed: Evaluation of the main nutritional parameters of raw materials by physicochemical analysis. Quantification of the quantity of micro / macroelements by atomic absorption techniques. Quantification of total phenols, antioxidant activity, kinetic activity and 50% IC of raw materials. Definition of the raw material absorption spectrum Study of the influence of lactic acid bacteria on the nutritional quality of quinoa and rice sourdough during fermentation In this sense, the following specific objectives were developed: Evaluation of the main nutritional parameters of sourdough by physicochemical analysis. Quantification of the amount of micro / macroelements, amino acids, aroma compounds, folic acid, lactic acid, chromatographic and spectophotometric techniques as well as determination of protein factions due to their molecular weight by SDS PAGE. V

6 Study regarding the improvment of quality and nutritional parameters in aglutenic functional products obtained through lactic sourdough fermentation quantification of total phenols, antioxidant activity, kinetic activity of the sourdough during fermentation process defining the absorption spectrum of quinoa and rice sourdough Obtaining an innovative functional product for people with celiac desease In order to achieve this objective, was pursued the experimental protocol from FIG. 2, having the following objectives: Valorization of sourdough fermented with lactic bacteria in an innovative functional product. studying and quantification of the main bioactive compounds from the sourdough and the final product. Evaluation of nutritional, physicochemical, microbiological and psycho-sensorial parameters of the final products (gluten muffins) Functional aglutenic muffins Evaluation of nutritional quality by GS-MS, LC / MS, AAA, atomic absorption,sds-page, spectrometry. Moisture Proteins Lipids Minerals Amino acids Folic acid Total phenols Antioxidant activity Lactic acid UV-VIS spectrum Sensory quality and flavor profile evaluation through GS / MC Color Flavor Taste Texture Overall appreciation Volatile compounds Evaluation of microbiological criteria FIG. 2. Experimental protocol for the characterization of bioactive compounds in the final product. VI

7 Maria Simona Chiș CHAPTER IV. RESULTS AND DISCUSSIONS 4.1. RESULTS AND DISCUSSIONS ON RAW MATERIALS This subchapter presents the results on the raw materials used for manufacturing the final product. The statistical analysis of these results was achieved by the Duncan test - the multiple comparison method. By statistically centralizing the data obtained in this part of the thesis, it was noted that quinoa flour had a content of minerals, amino acids, proteins, total phenols, antioxidant activity statistically different (p <0.05) compared to other materials raw materials used in the manufacture of the final product RESULTS AND DISCUSSIONS ON THE SOURDOUGH In this subchapter, the results obtained in the second general objective were disseminated, namely studying the qualitative and nutritional influence of quinoa and rice sourdough fermented with lactic acid bacteria. For the statistical interpretation of the obtained results, the Duncan test was used - the mutiple comparison method as well as the correlation coefficient Person to highlight the intensity of the link between two or more variables. In order to achieve this goal, four types of sourdough were produced, such as: quinoa sourdough inoculated with Lactobacillus plantarum ATCC 8014, quinoa sourdough subject to spontaneous microflora fermentation, rice sourdough inoculated with Lactobacillus spicheri DSM and rice sourdough fermented by spontaneous microflora. Comparing the spontaneous fermentation with a fermentation controlled by lactic acid bacteria, it was observed that there are statistical differences between the two types of fermentations, reflected by the amount of bioactive compounds: folic acid, lactic acid, radical scavening activity, total phenol content, amount of micro-macro elements, amino acids, aroma compounds, protein fractions. The main effect of the presence of lactic acid bacteria in sourdough is the acidification of the matrix, the process being caused by the conversion of fermentable carbohydrates mainly into lactic, acetic, formic or ethanolic acids. At the ph value of 4, a noticeable effect of protein solubilization is observed, which directly influences the proteolysis process and also the interaction between proteins and other partner molecules. With the decrease of the ph sourdoughs, the TTA (total titratable acidity) and the lactic acid content increase, thus creating an optimal environment for the proteolytic system development of lactic acid bacteria, which in turn degrade the release of assimilable compounds by lactic acid bacteria, thus ensuring their development. VII

8 Study regarding the improvment of quality and nutritional parameters in aglutenic functional products obtained through lactic sourdough fermentation The Pearson correlation coefficient between cell growth and ph of quinoa sourdough fermented with Lactobacillus plantarum (r = 0.997) indicates an extremely strong relationship between these two values, confirming once again that cell growth is maily strongly influenced by ph. Also, the correlation coefficient r = indicates a strong relationship between cell growth and ph of the rice sourdough fermented with Lactobacillus spicheri. Mineral content of the sourdough is influenced by the presence of phytic acid in quinoa flour and rice, antinutrient that binds metallic Fe, Ca, Mg and Zn ions, reducing their bioavailability. By fermentation with lactic acid bacteria, the amount of phytic acid is reduced, and thus mineral compounds change its bioavailability. During the spontaneous quinoa sourdough fermentation, there is a statistically significant increase (p <0.05) of the flavor compounds which give to the final product a less pleasant note, such as acetic acid, benzoic acid, 2-3-butanediol. In the quinoa sourdough, during controlled fermentation, there is a significant increase in flavor compounds, which gives it a finely-flavored to the final product: carvone, limonene, acetophone. In the spontaneously fermented rice sourdough, at the end of the fermentation process, there is a statistically significant increase in less pleasant flavor compounds such as benzoic acid, acetic acid, 2-3 butanediol and ethanol. In the case of the Lactobacillus spicheri (Sp) sourdough, there is a statistically significant increase in fruity flavoring compounds, mint, citrus, must, floral, almond 4.2. RESULTS AND DISCUSSIONS REGARDING THE AGLUTENIC MUFFINS This subchapter presents the results regarding the final products made with quinoa sourdough, respectively with rice sourdough, inoculated and by spontaneous fermentation at three different moments of fermentation: zero time, time 12 hours fermentation, respectively after 24 hours of fermentation. Following statistic analysis by the multiple comparison method, there are statistically significant differences (p <0.05) of the amino acid, mineral, aromatic content between the aglutenic muffins produced with quinoa sourdough at zero fermentation time compared to the muffins produced with fermented sourdough after 12 or 24 hours respectively. This is justified by the different content in the bioactive compounds of quinoa sourdough, with correlation coefficients indicating strong relationship between the bioactive compounds content of the sourdough and the final products. Thus, between the folic acid content of quinoa sourdough with Lp and the produced muffins there is a correlation coefficient of r = between the basic amino acid, acid, ɣ-aminobutyric, aliphatic and aromatic content of the quinoa inoculated sourdough and the finished products there is a correlation coefficient of 0.001, 0.995, 0.981, 0.987, VIII

9 Maria Simona Chiș Regarding the content of muffins manufactured with spontaneously fermented rice sourdough or with Lactabacillus spicheri (Sp) sourdough, there are statistically significant differences (p <0.05) between the amino acid, mineral and aroma content, results that could be justified through the different amino acid content of rice sourdoughs. Correlation coefficients of 0.991, 0.996, 0.989, 0.997, 0.999, indicate strong relationship between the basic amino acids, acids, ɣ-aminobutyric, aliphatic and aromatics of Sp fermented rice sourdough and final products. Also, it has been observed an extremely strong relationship between the acidity of the final product and the development of total yeast and mold (NTDM), justified by the formation of lactic acid, acetic acid, as well as possible antifungal peptide, during controlled fermentation. Thus, there is a correlation coefficient of between the acid content and NTDM for the aglutenic muffin produced with quinoa sourdough with Lp, and also, there is a correlation coefficient of between the acid content and NTDM at the aglutenic muffins produced with the rice sourdough with Sp. CHAPTER V CONCLUSIONS AND FUTURE RESEARCH DIRECTIONS 5.1. CONCLUSIONS REGARDING THE RAW MATERIALS As a result of the analyzes carried out in the first research section of the chemical composition of raw materials used in order to produce aglutenic functional muffins, the following aspects were highlighted: Quinoa flour is the raw material with a statistically significant difference in protein, amino acids, macro and micro elements, total phenols, antioxidant activity content compared to the other raw materials. Quinoa flour has a protein content of 14.5%, lysine content is 3.1g/100g of protein, histidine 2.4g/100g of protein, of tryptophan 0.9 g/100g of protein, a total phenolic content of 451 μg GAE / 100 g, a radical scavening activity of 92.05% and a Ca content of mg/100g, Mg mg/100g and K mg/100g. Rice flour it is characterized by a protein content of 8.5%, lysine of 1.2 g/100g protein, histidine 1.2 g/100g protein, tryptophan 0.6g/100 g of protein with a total phenol content of 258 μg GAE/100 g, a radical scavening activity of 83.58% and a Ca content of mg/100g, mg/100g Mg, and K of mg / 100g CONCLUSIONS REGARDING THE SOURDOUGHS Following the analyzes carried out under the second direction of research,,study regarding the qualitative and nutritional influence of the lactic acid bacteria on quinoa and rice sourdoughs during fermentation, the following aspects have been noted: IX

10 Study regarding the improvment of quality and nutritional parameters in aglutenic functional products obtained through lactic sourdough fermentation A parameter that undergoes changes during fermentation is cell growth of Lp and Sp. Thus, starting at a concentration of log 3,2 cfu/ml after 24 hours of fermentation, a concentration of log 13.5 cfu/ml was reached, which confirms the ability of Lp to displace the initial flour microflora and dominates during fermentation. In the case of the rice sourdough with Sp, from an initial concentration of 3.2 log cfu/ml was reached after 24 hours of fermentation a final concentration of 12.4 log cfu/ml. Although Sp was isolated from rice sourdough, it seems that its adaptability is lower compared to that of Lp, which again shows its ability to adapt to the most diverse environments. With increasing fermentation time, in the case of inoculated sourdoughs compared to sourdoughs fermented by spontaneous microbiota, there is a statistically significant increassement in the content of the following compounds: minerals, amino acids, aroma compounds, total phenols. The increasing content of amino acid is justified by protein hydrolysis by Lp and Sp due to their proteolytic system, which ends with the formation of flavor compounds and protein fractions identified by SDS PAGE (sodium dodecyl sulfate polyacrylamide gel electrophoresis). X 5.3. CONCLUSIONS REGARDING THE AGLUTENIC MUFFINS Regarding conventional foods, functional gluten-based technology has an important additional focus on identifying, quantifying and optimizing bioactive components with a functional role as well as monitoring them in the final product. Following the achievement of the third overall objective,,,obtaing an innovatve aglutenic functional product for people with special needs, the following conclusions were drawn: Muffins are natural products, without the addition of synthetic dyes, preservatives, flavors or various premixes, with a high nutrient content and are predominantly intended for celiacs as well as for people who are not intolerant to gluten. As a result of the analyzes carried out in the third research direction, it was noted that the addition of quinoa sourdough inoculated with Lp and Sp, determines a statistically significant increase of the minerals, amino acids content as well as flavor compounds. After interpreting the results of the psycho-sensory analysis, it was concluded that consumers' preference is directed towards the aglutenic muffin with quinoa sourdough with inocul of Lp, after 24 hours of fermentation. Regarding the microbiological quality of the aglutenic muffins with lactic acid bacteria, the results obtained are within the limits set by the Romanian Official

11 Maria Simona Chiș Monitor, Part I, No: 435 / on microbiological and hygiene standards on bakery products, cereal flours and derivates. Regarding the possibility of producing these muffins in a food industry unit, it can be concluded that the technological flow does not require special facilities besides the conventional products and that the working parameters can be the same. FUTURE RESEARCH DIRECTIONS New research ideas can be directed in the following directions: 1. Identification and study of antioxidant peptides resulting from LAB fermentation by a new RP-HPLC (Reverse Phrase High Performance Liquid Chromatography) technique, peptides that may have positive effects on human health, according to the latest studies 2. Influence of the initial consistency of sourdoughs on the degree of cellular development of lactic acid bacteria and implicitly on sourdoughs functional compounds. 3. Influence of certain parameters: temperature, time, degree of extraction of quinoa and rice flour, addition of carbohydrates on cell growth and on bioactive compounds. 4. Performing a comparative study of nutrients content from natural sources (quinoa and rice) and synthetic ones. 5. Manufactering new aglutenic products but also conventional ones by using sourdoughs with lactic acid bacteria and following their influence on the functional compounds of the final product. 6. Study of the influence of quinoa and rice sourdoughs on the textural quality of functional aglutenic products. THE THESIS ORIGINALITY AND INNVATIVE CONTRIBUTIONS The original elements of the thesis are: 1. Study of the influence Lactobacillus spicheri DSM (Leibniz Institute - German Collection of Microorganisms and Cell Cultures) strain on functional compounds of rice sourdough and on the final products. 2. Study of the influence of the Lactobacillus Plantarum ATCC (American Type Culture Collection) strain 8014 on quinoa sourdoughs functional compounds and on the final products 3. Adaptability study of the ATCC 8014 Lp strain in quinoa sourdough, being the first adaptability study of this strain in a quinoa flour medium. 4. Use of advanced techniques (GS / MS, HPLC coupled with LC / MS, SDS-page, atomic absorption spectrometry) to establish the detailed chemical composition of the sourdoughs and on the final products. 5. Identification of bioactive compounds in quinoa flour and rice with applicability in the food industry in order to obtain a special nutritional product. XI