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Synthesis of taste-active #-glutamyl dipeptides during sourdough fermentation by Lactobacillus reuteri Cindy J. Zhao, and Michael G. Gänzle J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.6b02298 • Publication Date (Web): 17 Sep 2016 Downloaded from http://pubs.acs.org on September 18, 2016
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Journal of Agricultural and Food Chemistry
Synthesis of Taste-Active γ-Glutamyl Dipeptides during Sourdough Fermentation by Lactobacillus reuteri Cindy J Zhaoa) and Michael G Gänzle* a,b)
a)
University of Alberta, Department of Agricultural, Food and Nutritional Science, Edmonton, Canada
b)
Hubei University of Technology, College of Bioengineering and Food Science, Wuhan, P.R. China
*Corresponding author footnote 4-10 Ag/For Centre Edmonton, AB T6E2P5, Canada Phone:+1 780 492 3634 Fax: + 780 492 4265 Email:
[email protected] 1 ACS Paragon Plus Environment
Journal of Agricultural and Food Chemistry
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Abstract:
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This study aimed to assess whether peptides influence the taste of sourdough bread. γ-
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Glutamyl dipeptides with known kokumi taste threshold, namely γ-Glu-Glu, γ-Glu-Leu, γ-Glu-
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Ile, γ-Glu-Phe, γ-Glu-Met and γ-Glu-Val, were identified in sourdough by liquid
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chromatography-tandem mass spectrometry in MRM mode. γ-Glutamyl dipeptides were found in
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higher concentrations in sourdough fermented with L. reuteri when compared to the chemically
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acidified controls. Proteolysis was an important factor for generation of γ-glutamyl dipeptides.
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Sourdoughs fermented with 4 strains of L. reuteri had different concentrations of γ-Glu-Glu, γ-
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Glu-Leu and γ-Glu-Met, indicating strain-specific differences in enzyme activity. Buffer
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fermentations with L. reuteri confirmed the ability of the strains to convert amino acids to
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γ-glutamyl dipeptides as well as the strain-specific differences. Sensory evaluation of bread
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revealed that sourdough bread with higher concentrations of γ-glutamyl dipeptides ranked higher
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with respect to the taste intensity when compared to regular bread and type I sourdough bread.
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Sourdough breads fermented with L. reuteri LTH5448 and L. reuteri 100-23 differed with respect
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to the intensity of the salty taste; this difference corresponded to a different concentration of γ-
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glutamyl dipeptides. These results suggest a strain-specific contribution of γ-glutamyl dipeptides
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to the taste of bread. The use of sourdough fermented with glutamate and kokumi peptide
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accumulating lactobacilli improved the taste of bread without adverse effect on other taste or
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quality attributes.
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Keyword: γ-glutamyl dipeptides, kokumi, sourdough, Lactobacillus reuteri
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Journal of Agricultural and Food Chemistry
INTRODUCTION
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Kokumi active compounds impart mouthfulness, complexity and continuity of taste.1,2 In
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contrast to compounds eliciting the six basic tastes sweet, sour, bitter, salty, umami, and
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oleogustus, kokumi compounds do not interact with taste receptors but enhance the perception of
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other taste compounds.3 Kokumi taste activity relates to the interaction of the calcium-sensing
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receptor CaSR.4 The kokumi peptides γ-Glu-Cys-Gly, γ-Glu-Val, γ-Glu-Ala, γ-Glu-Cys, and γ-
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Glu-Val-Gly are CaSR agonists and modulate signal transduction from taste receptors.4, 5
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Kokumi peptides were isolated from ripened cheese, beans, yeast extract, and soy
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sauce.1,2,6,7,8,9. Addition of γ-Glu-Val-Gly in chicken soup and reduced-fat cream significantly
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increases the thickness of taste and the intensity of aftertaste.5 Omission and reconstitution
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experiments in aqueous solution and in a cheese matrix confirmed that specific γ-glutamyl
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dipeptides impart the kokumi sensation.7,9 Glycopeptides and γ-glutamyl tripeptides also have
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kokumi activity.1,10,11,12 In contrast, α-glutamyl dipeptides do not have kokumi activity but may
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impart umami taste.
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Formation of kokumi peptide in soy sauce and cheese has been attributed to microbial
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activity, or enzymes present in raw milk. Penicillium roquefortii used in cheese ripening exhibits
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GGT activity;13 GGT activity was also characterized in Bacillus spp. used in fermentation of soy
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sauce.14 Analysis of glutamyl dipeptides in Parmesan and Gouda cheese revealed that the level of
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kokumi active γ-glutamyl dipeptides increased during ripening;715 the increase in Parmesan was
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attributed to GGT activity from raw milk rather than microbial enzyme activities.15 Different
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from α-glutamyl peptides, γ-glutamyl peptides were resistant to hydrolysis by peptidases.16 The
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proteolysis of casein only yields α-bonds; therefore, the presence of γ-glutamyl peptides can be
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attributed to microbial activity.1 Some studies indicated that some strains of Lactobacillus spp. 3 ACS Paragon Plus Environment
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produce γ-glutamyl peptide from free amino acids but their contribution remains unclear.15,17,18
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The microbial formation of kokumi-active γ-glutamyl peptides in fermentation of cheese and soy
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sauce implies that these peptides may also be formed in lactic fermentation of other protein-rich
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substrates, i.e. meat and cereal fermentations.
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Lactic acid bacteria are increasingly used in bread production; sourdough products are a
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small but fast growing segment of the baking improver market in North America. Sourdough
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fermented with lactic acid bacteria and yeasts has traditionally been used as leavening agent.19
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The current use of sourdough in industrial applications, however, employs sourdough mainly as
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baking improver to replace additives, and to improve bread quality.19 Effects of sourdough on
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bread quality are based on the activity of cereal enzymes during fermentation, acidification, and
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the microbial conversion of carbohydrates, lipids, and proteins.20,21
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Amino acids and their metabolites play a key role for improved bread quality.22 Lactate
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and glutamate are among the most prominent taste active compounds in bread. Glutamine
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conversion to glutamate by glutamate decarboxylase positive lactobacilli enhances the umami
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taste of bread, and addition of sourdough with high levels of glutamate allows reduction of NaCl
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levels without compromising bread quality. 23 The contribution of peptides to the taste of
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(sourdough) bread, however, remains unknown. It was therefore the aim of this work to
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quantitate γ-glutamyl dipeptides from sourdough fermented by different strains of L. reuteri, to
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determine whether lactobacilli contribute to the formation of γ-glutamyl dipeptides, and to
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provide an initial assessment of their role by sensory analysis of bread with different
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concentrations of γ-glutamyl dipeptides.
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MATERIALS AND METHODS
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Strains and growth conditions 4 ACS Paragon Plus Environment
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L. reuteri TMW1.106, LTH5448, 100-23, and 100-23∆gadB24 were grown in modified
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de Man, Rogosa and Sharpe Medium (mMRS) at 37°C. L. sakei Ls8 was grown in mMRS at
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32°C. Inocula for fermentation were prepared by harvesting of cells from overnight cultures in
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mMRS. Cells were washed twice in sterile tap water and resuspended in tap water to the initial
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culture volume. Inocula used for bread production were grown in 10% food grade wort for 24 h
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(CBW Munich pure malt extract, BRIESS Malt & Ingredients Co., Chilton, USA). Strains were
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subcultured twice and used for sourdough fermentation without washing.
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Materials and chemicals
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Transglutaminase was obtained from Ajinomoto (Tokyo, Japan). The dipeptide standards
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γ-Glu-leu, γ-Glu-Ile, γ-Glu-Glu, γ-Glu-Met, γ-Glu-Val, and γ-Glu-Phe were obtained from
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United BioSystems Inc (Herndon, VA, USA). Fungal protease from Aspergillus oryzae was
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obtained from Sigma-Aldrich (St. Louis, MO, USA). Food grade Flavourzyme, a combination of
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endoproteases and endopeptidases from Aspergillus oryzae with > 500 U / g, was obtained from
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Lallemand Baking solutions (Anjou, QC, Canada). Rye malt flour was kindly provided by
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Laihian Mallas (Laihia, Finland); whole rye flour and vital wheat gluten were obtained in a local
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supermarket.
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Sourdough fermentation with different ingredients, and with different strains of L. reuteri
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In order to understand the effect of the raw material, sourdoughs were fermented by
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L. reuteri 100-23 and L. reuteri 100-23∆gadB with or without 2.31 µL / g fungal protease,23 with
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or without 10UI/g microbial transglutaminase25 , and with rye malt or rye flour. Samples were
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taken after 0 h, 48 h, or 96 h of fermentation. The pH and cell counts were determined in fresh
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sourdough. Freeze-dried sourdoughs were used to quantitate free amino nitrogen and γ-glutamyl
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dipeptides as described below. All fermentations were carried out in two biological replicates 5 ACS Paragon Plus Environment
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and samples were analysed in duplicate. Results are reported as means ± standard deviation of
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duplicate independent experiments.
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To determine whether peptide conversion is strain specific, four different strains of L.
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reuteri (4 ml) were mixed with 0.5 g rye malt and 0.5 g vital gluten and fermented at 37°C for 24,
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48, 72 and 96 h. Chemically acidified dough was prepared by acidification with acetic acid:
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lactic acid (1:4, v/v) to a final dough pH of 3.5±0.25.
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Synthesis of γ-glutamyl peptides during buffer fermentation
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L. reuteri 100-23 and L. reuteri LTH5448 were grown in mMRS overnight and washed
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twice with autoclaved tap water. An aliquot of this culture was mixed with buffer containing
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5 g/L of maltose and 10 mmol/L of lysine, methionine, glutamine, glutamate, leucine, isoleucine,
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phenylalanine, and valine at 37°C at pH 6.5. Samples were collected after 0, 24, 48, 72 and 96 h
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for LC-MS/MS analysis of γ-glutamyl dipeptides in the culture supernatant. Controls inoculated
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with strains and maltose but without addition of amino acids, and uninoculated controls with
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maltose and amino acids were also analysed. The cell count and pH were determined on each
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sample. Experiments were carried out in two biological replicates and samples were analyzed in
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duplicate.
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Bread baking
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The bread used for evaluation by a consumer panel was baked in the food laboratory of
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the Department of AFNS at the University of Alberta. Cultures of L. reuteri (50 ml) were mixed
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with 50 g white wheat flour and fermented at 37°C for 24 h. The sourdough was propagated by
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addition of 75 g rye malt flour, 75 g wheat gluten, 0.75 g Flavourzyme, and 350 ml water, and
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fermented at 37°C for 72 h. A control sourdough was prepared by fermentation of wheat flour
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with L. sakei for 24 h at 32°C. This sourdough was used because the pH and the lactate
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concentration was comparable to sourdoughs fermented with L. reuteri; however, proteolysis and
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the formation of taste-active amino acids or peptides was limited by the short fermentation
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time.21,28 Samples were collected immediately for pH, cell count and high resolution melting
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curve –quantitative PCR (HRM-qPCR) analysis. L. sakei grew to cell counts of 109 cfu/g after 24
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h of fermentation. Sourdough was freeze dried and stored at room temperature for use in baking.
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Bread dough was prepared with 95% white wheat flour, 5% dried sourdough, 2% sugar, 2%
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yeast, 1.5% or 2% salt, and 70% water (ingredient dosage calculated on the basis of total flour
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weight), and mixed in a spiral kneader (Kitchen Aid K45SS, Hobart Co. Troy, OH, USA) for 5
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min. After a dough rest for 2 h at 30 °C in a proofer, the bread was shaped by hand and placed
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for 1 h in a proofer (Res-Cor, Crescent Metal Products Inc, Cleveland, OH, USA). Bread was
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baked in a multi-deck oven (Bakers Pride, Lachine, QC, Canada) with forced air at a temperature
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of 210 °C for 25 min. The bread was cooled down for 2 h at room temperature, packed in sealed
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polyethylene bags, and stored frozen at -18°C for sensory evaluation.
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DNA isolation and HRM qPCR identification
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The identity of the sourdough fermentation microbiota with the inoculum was verified by
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HRM-qPCR.26 In brief DNA was isolated from 1.0 mL fresh sourdough or overnight cultures of
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L. reuteri LTH5448 and 100-23, and L. sakei LS8 grown in mMRS broth using a DNeasy Blood
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and Tissue kit according to the instructions of the manufacturer (Qiagen, Mississauga, ON,
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Canada). HRM-qPCR (Rotor-GeneQ, Qiagen, Mississauga, ON, Canada) was used to amplify
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bacterial 16S rDNA.
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Sensory consumer panel
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The sensory studies were reviewed for their adherence to ethical guidelines and approved by the Research Ethics Board at the University of Alberta.
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The frozen bread was thawed 24 h at room temperature before sensory analysis. The
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bread crust was cut off and the breadcrumb was cut into 1 cm3 pieces. The samples were placed
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in separate covered petri dishes labeled with 3-digit random numbers and 3 pieces were
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presented to the panelists. Sensory evaluation was performed in the sensory testing laboratory at
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the Department of Agricultural, Food and Nutritional Science, University of Alberta.
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The panelists were recruited randomly at the Agricultural & Forestry Centre, University
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of Alberta. The number of the male and female panelist was about equal. Most of the panelists
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(78%) were 18-29 years old. A majority of panelists consumed bread more than 2-3 times per
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week and indicated that they like bread. Over 60% of the panelists thought taste is the most
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important attribute compared to flavor, texture and color.
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The panelists received encoded samples and questionnaire, as well as instructions for the
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evaluation of samples. Samples were randomly assigned to each panelist. The samples were
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presented to the assessors blind, so that the panelists did not know which sample they were
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evaluating. Water was provided to cleanse the palate between samples. The preference and
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paired comparison on salty taste were used for 1.5% and 2% salt level bread as described.23 The
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number of correct responses and the number of total responses were counted. The Just About
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Right (JAR) test was used to evaluate the saltiness and aftertaste of the bread fermented by L.
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reuteri 100-23 and L. reuteri LTH5448 at 2% salt level. The frequency of each category was
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calculated. The intensity of saltiness of L. reuteri 100-23, L. reuteri LTH5448, L. sakei LS8 and
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regular bread at 1.5% salt level was evaluated by ranking test. The sum of the rank for each
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sample was calculated and the result was analyzed by Friedman test.27 8 ACS Paragon Plus Environment
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Journal of Agricultural and Food Chemistry
Free amino nitrogen (FAN)
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Free amino nitrogen (FAN) content of the SDS-soluble fraction in different samples was
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measured by the ninhydrin method. Freeze-dried samples (50 mg) were suspended in 1 mL
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sodium phosphate buffer (200 mmol L-1) and incubated at 23°C with agitation (250 rpm) for 1 h.
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Solids were removed by centrifugation for 10 min at 10,000 x g; 10 µL of supernatant was mixed
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with 190 µL of phosphate buffer and 100 µL of ninhydrin solution (5.0 g Na2HPO4, 6.0 g of
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KH2PO4, 0.3 g of fructose and 0.5 g of Ninhydrin (Sigma-Aldrich, USA) in 100 mL of Milli-Q
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water at pH 6.7) and incubated at 100°C for 16 min. After cooling to room temperature for 20
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min, the sample was mixed with 500 µl of KIO3 solution (0.2 g KIO3 dissolved in 60 mL of
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distilled water and 40 mL of 96% ethanol) and the absorbance was measured at 570nm. Glycine
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solution with concentrations from 2.0 to 20.0 mg L-1 was used to establish a calibration curve.
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Quantitation of kokumi peptides by LC-MS/MS analysis
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Peptide quantitation was performed using a 1200 series HPLC unit and diode array
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detector (DAD) (Agilent Technologies, Palo Alto, CA, USA) connected to a 4000 Q TRAP LC-
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MS/MS System (MDS SCIEX, Applied Biosystems, Streetsville, ON, Canada). Peptides were
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separated on a Luna C18 RP-HPLC column (5 um, 250 mm X 4.6 mm, Phenomenex, Torrance,
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CA, USA) and detected from 190 to 400 nm. Mobile phase A consisted of 0.1% formic acid in
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Milli-Q water. Mobile phase B consisted of 0.1% formic acid in acetonitrile. Samples were
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eluted at a flow of 0.5 ml min-1 with the following gradient: 0-16 min, 100%A; 16-20 min, 100-
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95%A; 20-30 min, 95-75%A; 30-45 min, 75-65%A, 45-47 min 65-0%A; 47-57 min, 0%A; 57-
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67 min, 100%A; and re-equilibration time of 10 min.
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LC-MS/MS analysis was performed using atmospheric pressure electrospray ionization
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in positive mode. The protonated precursor ions and the dominant fragments of each of the six9 ACS Paragon Plus Environment
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kokumi peptides were optimized. The samples were detected and quantitated using multiple
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reaction monitoring mode (MRM). LC-MS/MS parameters for quantitation of the six kokumi
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peptides are shown in Table 1. The values for optimum ion source parameters were as follows:
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spray voltage 4 KV, collision energy 10, curtain gas 10, and declustering potential at 20 V. Data
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acquisition was interfaced to a computer workstation running Analyst 1.5 (Applied Biosystems,
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USA). External calibration standards (0.005 to 50 mg/L) of γ-glutamyl dipeptides were prepared
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at 30% (v/v) methanol in 0.1% aqueous formic acid. The limit of quantitation is 0.5 mg/L.
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Statistical analysis
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Statistical analysis of concentration of free amino nitrogen of sourdough was performed
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by analysis of variance (ANOVA) with the procedure PROC GLM using the Statistical Analysis
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System V.9.2 (SAS Institute Inc., Cary, NC, USA). Values were considered significantly
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different at a 5% error level (P < 0.05). Analysis of concentrations of γ-glutamyl dipeptides was
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performed with repeated measurement using GLM procedure (PASW Statistics 18.0; IBM SPSS
194
Statistics, USA). P value of ≤ 0.05 with Tukey adjustment for multiple comparisons was
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considered statistically significant.
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RESULTS
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Characterization of sourdoughs
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Sourdough was fermented with different raw materials and with different strains for up to
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96 h. All colonies obtained from sourdoughs exhibited a uniform colony morphology matching
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the inoculum, indicating identity of fermentation microbiota with the respective inoculum. Strain
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identity was further confirmed with HRM-qPCR with template DNA from sourdoughs. Size and
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melting temperature (86.5°C) of amplicons obtained from sourdough matched the amplicons 10 ACS Paragon Plus Environment
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from L. reuteri (data not shown). Cell counts and pH of sourdoughs fermented with L. reuteri
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were comparable with results previously obtained with the same strains (Table S1).23 Cell counts
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and pH of sourdoughs fermented with different raw materials or different strains were not
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different. Chemically acidified doughs maintained a pH of 3.4-4.0 throughout the incubations
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and the cell counts in all of the chemically acidified controls were below 104 cfu g-1.
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Sensory evaluation of salty taste in different types of bread
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To understand the effect of different sourdough fermentations on the taste of bread, the
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taste of bread produced with sourdough that was fermented with L. reuteri for 72h was compared
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to bread produced with L. sakei and 24 h of fermentation. L. sakei produced equivalent levels of
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acidity as strains of L. reuteri and acidified the sourdough to a pH of 3.5 - 3.6 after 24 h of
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fermentation (data not shown). Bread produced with a straight dough process served as reference.
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The consumer panel (n=42) was asked to rank the salty taste intensity of the breads, which
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contained 1.5% salt level. The salty taste intensity of sourdough bread produced with L. reuteri
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100-23 and LTH5448 ranked significantly higher (rank sum 142 and 132, respectively) than
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regular bread (rank sum 67, P
