Real-Time Monitoring of Chemical Changes in Three Kinds of

Article Cite This: J. Agric. Food Chem. 2018, 66, 1479−1487

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Real-Time Monitoring of Chemical Changes in Three Kinds of Fermented Milk Products during Fermentation Using Quantitative Difference Nuclear Magnetic Resonance Spectroscopy Yi Lu, Hiroto Ishikawa, Yeondae Kwon, Fangyu Hu, Takuya Miyakawa, and Masaru Tanokura* Department of Applied Biological Chemistry, Graduate School of Agricultural and Life Sciences, The University of Tokyo, 1-1-1 Yayoi, Bunkyo-ku, Tokyo 113-8657, Japan S Supporting Information *

ABSTRACT: Fermented milk products are rising in popularity throughout the world as a result of their health benefits, including improving digestion, normalizing the function of the immune system, and aiding in weight management. This study applies an in situ quantitative nuclear magnetic resonance method to monitor chemical changes in three kinds of fermented milk products, Bulgarian yogurt, Caspian Sea yogurt, and kefir, during fermentation. As a result, the concentration changes in nine organic compounds, α/β-lactose, α/β-galactose, lactic acid, citrate, ethanol, lecithin, and creatine, were monitored in real time. This revealed three distinct metabolic processes in the three fermented milk products. Moreover, pH changes were also determined by variations in the chemical shift of citric acid during the fermentation processes. These results can be applied to estimate microbial metabolism in various flora and help guide the fermentation and storage of various fermented milk products to improve their quality, which may directly influence human health. KEYWORDS: fermented milk products, in situ quantitative monitoring, NMR

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acidic and alcoholic fermentation.12 It originates from the Caucasian mountains.13 The microbial populations in the kefir grain are Lactobacillus, Lactococcus, and yeast.14 Recently, studies on chemical compositions of fermented milk products have been carried out.15,16 In these studies, organic acids, acetaldehyde, and other compounds in fermented milk were extracted, separated, and then analyzed by liquid chromatography− mass spectrometry (LC−MS)15 and gas chromatography−mass spectrometry (GC−MS).16 In all of these steps (extraction, separation, and derivatization), even simple treatments could cause qualitative and quantitative changes to the original mixture. Nuclear magnetic resonance (NMR) is a highly quantitative and reproducible analytical technique.17 As a non-targeted method, NMR measurements do not require any separation or chemical modification, giving overall information regarding the chemical components of a mixture rapidly and directly. Therefore, NMR spectroscopy would allow us to perform in situ monitoring and quantitative analysis in real time. In the last few decades, NMR has been recognized as a powerful technique that has been widely used for metabolic description of foods, such as coffee,18−20 olive oil,21 juice,22 honey,23 and liquor as a fermented food.24,25 In addition, milk has been analyzed by NMR to identify and quantitate characteristic compounds26,27 and overview metabolite patterns for quality assessments.28−31 More recently, an in situ quantitative method using difference spectra between quantitative 1H NMR spectra and diffusionordered spectroscopy (DOSY) NMR spectra were developed

INTRODUCTION Potential health benefits have become key factors for consumers when making their food choices.1 Fermented milk products, which are a functional dairy food, have risen in popularity throughout the world because several lines of evidence have shown that fermented milk products exhibit many health benefits.2−4 The epidemiologic study also showed that common effects of fermented milks on an intestinal environment are increases in the counts of bifidobacteria and decreases in the counts of harmful bacteria.3 These effects may increase during fermentation;5 therefore, it is important that the fermentation processes are monitored and controlled. Among the various kinds of fermented milk products, Bulgarian yogurt, Caspian Sea yogurt, and kefir are widely consumed as health foods and for their sensory properties.4,6,7 Bulgarian yogurt is the most popular variety of yogurt in the world. It was invented in Bulgaria and is part of a heritage that dates back many centuries.6 Bulgarian yogurt is commonly made with two starter bacteria, Lactobacillus bulgaricus and Streptococcus thermophiles, which give the yogurt its characteristic thickness, acidity, taste, and aroma.8 S. thermophiles bacteria act first and prepare the proper environment for L. bulgaricus, which can begin to multiply and slowly turn the milk into yogurt.2 Caspian Sea yogurt, also known as Matsoni yogurt, is one of the yogurts that is cultured at room temperature.9 It originates from the Caucasus region between the Black Sea and Caspian Sea, a region famous for the longevity of the population.10 The microbiota of Caspian Sea yogurt is reported to be Lactococcus lactis subsp. cremoris (Streptococcus cremoris) and Acetobacter orientalis, which give it its uniquely viscous consistency.11 Kefir is a fermented milk product made with kefir grains, consists of lactic acid bacteria and yeasts, and is obtained by a combined © 2018 American Chemical Society

Received: Revised: Accepted: Published: 1479

November 14, 2017 January 16, 2018 January 17, 2018 January 17, 2018 DOI: 10.1021/acs.jafc.7b05279 J. Agric. Food Chem. 2018, 66, 1479−1487

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Journal of Agricultural and Food Chemistry for measuring the components in fermented milk products.32 In the present study, we applied the in situ quantitative NMR method to monitor the fermentation processes of different kinds of fermented milk products in real time. The obtained quantitative data could be used to guide the fermentation processes and storage of various fermented milk products, improving the quality of the fermented milk products, which can directly influence human health.

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and 1H−13C constant time heteronuclear multiple-bond correlation (CT-HMBC)] of the three fermented milk products (32 h fermentation at 40 °C for Bulgarian yogurt, 22 h fermentation at 25 °C for Caspian Sea yogurt, and 25 h fermentation at 25 °C for kefir) were measured at 4 °C on a Unity INOVA-500 spectrometer. The 13C NMR spectra were measured at 125.71 MHz. Dioxane was used as an external reference, and its chemical shift was set to 67.5 ppm. The parameters of the 13C NMR spectrum were as follows: number of

MATERIALS AND METHODS

Materials and Sample Preparation. The inoculums of Bulgarian yogurt (L. bulgaricus, S. thermophiles, and skim milk) (AFC Co., Ltd., Shizuoka, Japan) and Caspian Sea yogurt (S. cremoris FC, A. orientalis FA, and skim milk) (Fujicco Co., Ltd., Kobe, Japan), kefir grains (Lactobacillus casei strain Shirota, S. cremoris, Saccharomyces florentinus, and skim milk) (Nihon Kefir Co., Ltd., Fujisawa, Japan), and whole milk (Meiji Co., Ltd., Tokyo, Japan) were purchased at a local market. For fermentation monitoring, the inoculums of Bulgarian yogurt (1 g), Caspian Sea yogurt (6 g), and kefir grains (4 g) were each added to whole milk (1 L). The samples were immediately mixed with D2O to give final concentrations of 10% (v/v) and were then placed in 5 mm NMR tubes. The volumes of the samples were adjusted to 0.6 mL. A capillary containing 20% (v/v) 1,1,2,2-tetrachloroethane (Wako Pure Chemical Co., Ltd., Osaka, Japan), 80% (v/v) chloroform-d (Isotec, Inc., Tokyo, Japan), and 1 mg/mL chromium(III) acetylacetonate (Kanto Chemical Co., Ltd., Tokyo, Japan) was inserted into each NMR tube as the concentration standard.32 Each inoculum sample was prepared in triplicate from the same fermentation batch. NMR Spectroscopy. NMR measurements were performed at 40 °C (Bulgarian yogurt) or 25 °C (Caspian Sea yogurt and kefir) on a Unity INOVA-500 spectrometer (Agilent Technologies, Santa Clara, CA, U.S.A.) for the 1H quantitative NMR spectra (time for data collection of 10 min) and DOSY NMR spectra (time for data collection of 5 min). The prepared samples were placed in the NMR equipment, and the first sequential acquisition of the 1H quantitative and DOSY NMR spectra was carried out within 15 min. The collected spectra were defined as time 0. The fermentation was then performed on the NMR equipment at 40 °C for 32 h (Bulgarian yogurt), 25 °C for 22 h (Caspian Sea yogurt), and 25 °C for 25 h (kefir) without spinning or stirring. The 1 H NMR spectra were acquired at 5, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, and 32 h for Bulgarian yogurt, at 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 20, and 22 h for Caspian Sea yogurt, and at 11, 13, 15, 17, 19, 21, 23, 24, and 25 h for kefir during fermentation. At the end point, each sample was smelled and tasted similarly to the products prepared according to the standard protocols of the manufacturers. The 1H NMR spectra of the fermented milk products were measured at 499.87 MHz, and a HDO signal was suppressed by pre-saturation. The CH-β2 signal of lactose was used as an internal reference, and its chemical shift was set to 3.16 ppm based on the data of our previous study.32 For quantitation, the acquisition parameters were as follows: number of data points, 32 000; spectral width, 8000 Hz; acquisition time, 2.048 s; delay time, 15 s; and number of scans, 32. The delay time (d1) was determined with the spin−lattice relaxation time (T1) and the acquisition time (aq).

d1 ≥ 5T1 − aq The DOSY NMR measurements were carried out soon after each series of 1H NMR measurements was complete. The acquisition parameters were as follows: number of data points, 32 000; spectral width, 8000 Hz; acquisition time, 2.048 s; delay time, 15 s; number of scans, 16; diffusion delay, 0.4 s; total diffusion-encoding gradient pulse duration, 0.002 s; and gradient stabilization delay, 0.0003 s. The signals that overlapped with the signals of milk fats were quantitated using difference spectra that were obtained by subtracting the DOSY spectra from the quantitative 1H NMR spectra.32 For NMR signal assignments, 13C and two-dimensional (2D) NMR spectra [1H−13C heteronuclear single-quantum correlation (HSQC), 1 H−1H double-quantum filter correlation spectroscopy (DQF-COSY),

Figure 1. Quantitative 1H NMR spectra of (A) Bulgarian yogurt, (B) Caspian Sea yogurt, and (C) kefir during fermentation. HDO signals (4.4−4.8 ppm) were suppressed. 1480

DOI: 10.1021/acs.jafc.7b05279 J. Agric. Food Chem. 2018, 66, 1479−1487

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Journal of Agricultural and Food Chemistry

in our previous study32 was used to investigate the changes in the chemical composition over time. The integral value of 1,1,2,2-tetrachloroethane was compared to those of the compounds in the three fermented milk products to determine their concentrations. pH Measurements. Citrate−phosphate buffer (0.1 M) prepared with D2O was adjusted to each pH value (pH 4.0−6.8) using a Twin pH B-212 pH meter (Horiba, Kyoto, Japan). The 1H NMR spectrum of each buffer was recorded at 25 °C on a Unity INOVA-500 spectrometer. The standard pH titration curve was created by plotting chemical shifts of citric acid (CH-β2) against the pH values. The pH values during the fermentation process were calculated using the standard pH titration curve and the chemical shift values of citric acid (CH-β2).

data points, 64 000; spectral width, 31 422 Hz; acquisition time, 1.043 s; delay time, 2 s; and number of scans, 83 392. The 1H−1H DQF-COSY spectra were obtained by suppressing the water signal with pre-saturation, and the acquisition parameters were as follows: number of data points, 2048 (F2) and 512 (F1); spectral width, 5911 Hz (F1 and F2); acquisition time, 0.202 s; delay time, 2 s; and number of scans, 48. The 1H−13C HSQC spectra of the fermented milk were generated in the phase-sensitive mode with the following acquisition parameters: number of data points, 512 for 1H and 256 for 13C; spectral widths, 5498 Hz for 1H and 20 110 Hz for 13C; acquisition time, 0.186 s; delay time, 2 s; and number of scans, 80. The 1H−13C CT-HMBC spectra were measured in the absolute mode with the following parameters: number of data points, 4096 for 1 H and 512 for 13C; spectral widths, 5498 Hz for 1H and 27 643 Hz for 13 C; acquisition time, 0.402 s; delay time, 3 s; and number of scans, 80. NMR Signal Assignments and Data Processing. The preprocessing of the free induction decays (FIDs) and the subsequent Fourier transformations were performed by the program MestRe Nova 10.0 (MestRec, Santiago de Compostela, Spain). NMR signals were analyzed by comparison to our previous published data based on 2D NMR correlations, including NMR assignment data32 and composition data.33,34 The signals were then confirmed and assigned to the candidate compounds using the 2D NMR spectra. The quantitative method developed

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RESULTS AND DISCUSSION NMR Spectroscopic Analysis of the Three Fermented Milk Products. NMR spectral analyses of the three fermented milk products were carried out to monitor the chemical changes during fermentation on a solution-state NMR spectrometer. Figure 1 shows quantitative 1H NMR spectra of the three fermented milk products during fermentation. The initial sample (0 h) looked like a solution as a result of the mixture of milk and a small amount of each inoculum. On the other hand, the

Figure 2. Evolution of components in Bulgarian yogurt during fermentation. Data are means ± standard deviations (SD; n = 3). 1481


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Figure 3. Evolution of components in Caspian Sea yogurt during fermentation. Data are means ± standard deviations (SD; n = 3).

final samples (32, 25, and 22 h for Bulgarian yogurt, Caspian Sea yogurt, and kefir, respectively) were semi-solid. Although the states are different between initial and final samples, the observed spectra, especially the signal shapes, were quite similar. In addition, the 1H NMR spectra of the final samples were quite similar to those of the samples of a 100 mL scale-up fermentation. On the basis of 2D NMR analysis, spiking experiments, and peak assignments reported in a previous study,32 nine components were identified: α/β-lactose, α/β-galactose, lactic acid, citrate, lecithin, creatine, and ethanol. As shown in Figure 1B, signals indicative of galactose were not observed during fermentation of Caspian Sea yogurt, unlike Bulgarian yogurt and kefir, in which galactose was observed. As shown in Figure 1C, ethanol was only observed in kefir. Changes in Component Concentrations in the Three Fermented Milk Products. Our developed quantitation method32 was used to quantitate the concentrations of components in the three fermented milk products during fermentation. The integral values of signals were calculated on the difference spectra that were obtained by subtracting the DOSY spectra from the quantitative 1H NMR spectra. The 1H signal (5.83 ppm) of 1,1,2,2-tetrachloroethane was used as an external quantitative standard because it was separately observed from other signals. The nine detected compounds in the three fermented milk products were quantitated by comparing the integral values of their signals to that of the standard signal. The quantitative results are shown in Figures 2−4. Overall, the patterns of concentration changes were different among the three fermented milk products, even when the same compound was compared, especially citric acid and α/β-galactose. The concentration of α/β-lactose and citric acid were 146−185 mmol/L (50−63 g/L) and 3.1−4.5 mmol/L in the initial sample (0 h),

respectively. These values were not largely different from those in milk that was determined by a previous NMR study (47.8 g/L for α/β-lactose and 3.2 mmol/L for citric acid).27 The concentration of the nine detected compounds was hardly affected by comparison of the initial sample (0 h) to the sample after fermentation for about 10 h. Chemical Changes in Bulgarian Yogurt during the Fermentation Process. As shown in Figure 2, the quantities of lactic acid and citric acid did not vary greatly during the first 12 h or during the last 8 h but increased quickly between 12 and 24 h of fermentation. Continuous decreases in α/β-lactose were detected starting at 14 h, while the concentrations of α/βgalactose increased from 12 to 32 h. No obvious changes in the concentrations of lecithin or creatine were observed during fermentation. The α/β-lactose ratio observed during the fermentation process was 1:1.57 ± 0.014 (data are the mean ± standard deviation), which is slightly higher than that of the water solution (1:1.5 at 25 °C). The temperature for NMR measurements (40 °C) and the composition of organic compounds in Bulgarian yogurt products may influence the α/β-lactose ratio.35 The ratio was not largely changed during fermentation. L. bulgaricus and S. thermophiles are classified into lactic acid bacteria that generally decompose lactose to glucose and galactose and produce lactic acid by the glycolytic pathway and lactic acid fermentation (Figure 5). As shown in Figure 2, the increased production of lactic acid was observed at 14 h, which corresponds to the starting time of α/β-lactose consumption. Interestingly, the 1H NMR signals of glucose were not observed in the observed fermentation period (Figure 1A), which suggests that glucose from lactose degradation is immediately consumed in the glycolytic pathway. On the other hand, α/β-galactose began to increase at 12 h of fermentation. Furthermore, the 1482


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Figure 4. Evolution of components in kefir during fermentation. Data are means ± standard deviations (SD; n = 3).

increased amount of galactose (7.7 ± 0.7 mmol/L) during fermentation from 10 to 12 h was much higher than the amount of lactose consumption (2.2 ± 0.4 mmol/L) (Figure 2). This observation raises a possibility that galactose may be produced from multiple pathways, e.g., degradation of galactooligosaccharides,36 in addition to the hydrolysis of lactose. Citric acid was observed in the initial sample (0 h) in agreement with the citric acid content of milk. It is well-known that citric acid is a constituent of the tricarboxylic acid (TCA) cycle and is produced from glucose through the glycolytic pathway and the oxidative conversion of pyruvic acid into acetyl-CoA that is carried out by pyruvate dehydrogenase (Figure 5).37,38 The rapid

increase in the amount of citric acid is thought to be due to the mass production of pyruvic acid between 16 and 24 h (Figure 2), which does not contradict with the result of lactic acid that is also produced by pyruvate during lactic acid fermentation. However, the concentration of citric acid started to be elevated at 16 h, which is later than the starting point of lactic acid production. In addition, the production rate of lactic acid was 3-fold higher than that of citric acid. These observations suggest that pyruvic acid could be mainly used in the lactic acid fermentation. Lecithin is one of the major components of the phospholipid portion of the cell membrane39 and is a dietary source of 1483


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Chemical Changes in Kefir during the Fermentation Process. As shown in Figure 4, the concentration of lactic acid increased slowly between 11 and 17 h and from 23 to 25 h during fermentation and increased quickly between 19 and 23 h. Citric acid decreased sharply between 11 and 25 h and eventually disappeared. Lactose and galactose decreased substantially between 11 and 25 h of fermentation. The signals of ethanol were observed at 17 h, and the concentration of ethanol increased between 15 and 25 h of fermentation. The amount of lecithin and creatine did not vary to a large extent during fermentation. The α/β-lactose ratio observed in the present study was 1:1.55 ± 0.018 (data are the mean ± standard deviation), which is slightly higher than that of the water solution (1:1.5 at 25 °C) and is not largely changed during fermentation. Kefir grain mainly contains L. casei, S. cremoris, and S. florentinus in addition to a mixture of proteins, lipids, and sugars. Lactose could be decomposed to produce glucose and galactose by lactic acid bacteria as well as in Bulgarian yogurt and Caspian Sea yogurt. The metabolic pathway of yeast (Figure 5),46 which includes an alcohol fermentation pathway, is different from that of lactic acid bacteria. Yeast begins to work after the pH becomes sufficiently low, eventually producing ethanol by consumption of glucose. The quantitative results show that the similar chemical changes of lactic acid and lactose observed in Bulgaria yogurt and kefir were due to the action of Lactobacillus and Streptococcus, while ethanol was produced by yeast. This is the characteristic of kefir that makes it different from the other two kinds of yogurt. The glucose fermentation of yeast was monitored by NMR spectroscopy.47 As the reaction proceeds, the glucose signal was decreased, while the intensity of ethanol signals was increased, which corresponds to the results of kefir fermentation in the present study. The citric acid level decreased during the fermentation of kefir by the citric acid metabolism. The consumption rate of citric acid was the fastest among three fermented milk products in this study. On the other hand, production of citric acid in kefir may be less than those in other fermented milk products because pyruvate is also consumed by ethanol fermentation in addition to lactic acid fermentation (Figure 5). Kefir has unique and complex probiotic properties in which kefiran often functions as a promising compound. Kefiran is known to be a water-soluble EPS produced in kefir grain during fermentation that has anticancer, antiinflammatory, and hypocholesterolenic effects and can aid in managing the immune system and lowering blood pressure.48 Decreased galactose levels during kefir fermentation (Figure 4) suggest the production of kefiran because it is a galactose-rich polysaccharide. Relative Changes in Fatty Acids during the Fermentation Processes. To investigate the changes in the composition of fatty acids during the fermentation processes, the step-by-step integral values of the signals at 0.76 ppm were calculated (Figure 6). Fatty acids are naturally found in dairy products, and trans fatty acids are associated with biological effects, such as increased risk of coronary heart disease, which may directly affect human health.49 Furthermore, fatty acid synthesis, which occurs in the cytoplasm of the cell, is the process by which fatty acids are produced from acetyl-CoA and NADPH by enzymes.50 In this study, integrals of fatty acid signals did not change substantially during the fermentation processes, which indicated that the concentrations of NMR-detected fatty acids did not vary very much during fermentation. pH Changes in the Three Fermented Milk Products during Fermentation. Figure 7A shows the relationship between

Figure 5. Schematic diagram of major metabolic pathways during the fermentation of Bulgarian yogurt (pink arrows), Caspian Sea yogurt (cyan arrows), and kefir (orange arrows). The thickness of the arrow represents the pseudo-rate of metabolic flow. The bacterial strains most common in Bulgarian yogurt are L. bulgaricus and S. thermophiles. Caspian Sea yogurt is composed of S. cremoris and A. orientalis. The microbial populations in the kefir grain are L. casei, S. cremoris, and S. florentinus. EPS means exopolysaccharide.

choline, which can be converted into acetylcholine, an organic compound that functions as a neuromodulator in the brain.40 Creatine can be phosphorylated to form phosphocreatine, which is recognized as a kind of energy buffer in skeletal muscles and in the brain.41 These nutritional compounds were hardly affected during the fermentation of Bulgarian yogurt. Chemical Changes in Caspian Sea Yogurt during the Fermentation Process. As shown in Figure 3, the amount of lactic acid did not vary substantially during the first 12 h or the last 7 h but increased rapidly between 12 and 15 h of fermentation. The concentration of citric acid increased quickly between 12 and 14 h and then decreased sharply between 14 and 18 h of fermentation. Continuous decreases in α/β-lactose were observed between 9 and 20 h. The quantities of lecithin and creatine did not vary substantially during fermentation. The α/β-lactose ratio observed in the present study was 1:1.64 ± 0.018 (mean ± standard deviation), which is slightly higher than that of the water solution (1:1.5 at 25 °C). The ratio was not largely changed during fermentation. Unlike in Bulgarian yogurt, signals from galactose were not observed in Caspian Sea yogurt during fermentation. Galactose is incorporated into the cells by phosphoenolpyruvate:carbohydrate phosphotransferase system of S. cremoris.42 The system could be one of the reasons why the consumption of galactose during the fermentation of Caspian Sea yogurt was much faster than that in Bulgarian yogurt. Caspian Sea yogurt produces exopolysaccharide (EPS) during fermentation to become more viscous,11 and because galactose is a building block of EPS, it may be consumed and used in EPS production during the fermentation of Caspian Sea yogurt (Figure 5).43 The increase in citric acid between 12 and 14 h is likely caused by the mass production of pyruvate during lactic acid fermentation, whereas citric acid was decreased between 14 and 18 h. Lactic acid bacteria are also able to convert citric acid to some end products, such as diacetyl and acetaldehyde, which contribute to the quality of the fermented foods as a result of their distinct aroma properties.44,45 The metabolic flow of citric acid seems to be converted from production to degradation when lactic acid fermentation was terminated at 15 h. This metabolic change was not observed in Bulgarian yogurt, which may be affected by differences in microbial compositions between Bulgarian yogurt and Caspian Sea yogurt. 1484


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Figure 6. Relative changes in fatty acid levels during the fermentation of the three fermented milk products. Data are means ± standard deviations (SD; n = 3).

Figure 7. (A) Relationship between the chemical shift of citric acid and pH of a citrate−phosphate buffer (25 °C). The CH-β2 (3.16 ppm) signal of lactose was used as an internal reference. (B) 1H chemical shift changes of citrate in the three fermented milk products during fermentation. (C) pH changes in the three fermented milk products during fermentation. The blue line represents the chemical shifts of citric acid and the pH values calculated from the NMR spectra during the fermentation of Caspian Sea yogurt. The purple line represents the pH values measured by a pH meter during the fermentation of Caspian Sea yogurt. The red line represents the chemical shifts of citric acid and the pH values calculated from the NMR spectra during the fermentation of Bulgarian yogurt. The light green line represents the chemical shifts of citric acid and the pH values calculated from the NMR spectra during the fermentation of kefir.

study is the first application of the pH measurements from NMR spectra during fermentation of yogurt. The pH levels during the fermentation of Bulgarian yogurt and kefir were also calculated from NMR spectra (Figure 7C). This method provides a real-time, non-destructive way to confirm pH during fermentation of yogurt, which is required for quality control of yogurt. In conclusion, this study applied our newly developed quantitative NMR method for monitoring chemical changes in various fermented milk products during fermentation. The acquisition of quantitative 1H NMR spectra and DOSY NMR spectra is easy and quick, and the preparation of samples in organic solvent is simple, both of which are important advantages to this

the chemical shift of citric acid and pH of a citrate−phosphate buffer. Figure 7B shows the changes in the 1H chemical shift of citrate in the three fermented milk products during fermentation. The pH changes in Caspian Sea yogurt during fermentation were observed by two different methods. The pH values were first calculated by the chemical shifts of citric acid signals in the 1H NMR spectra during fermentation (panels A and B of Figure 7). Then, the pH values calculated from the NMR spectra were compared to the values determined by a pH meter (shown in Figure 7C). These results revealed that the pH values calculated during the fermentation of Caspian Sea yogurt were consistent with those measured by the pH meter. The present 1485


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or cholesterol fractional synthesis rates relative to milk in hyperlipidemic men: A randomized controlled trial. BMC Complementary Altern. Med. 2002, 2, 1. (8) Karagül-Yüceer, Y.; Drake, M. Sensory analysis of yogurt. In Manufacturing Yogurt and Fermented Milks, Chandan, R. C., Ed.; Blackwell Publishing: Ames, IA, 2016; Chapter 16, pp 265−276, DOI: 10.1002/9780470277812.ch16. (9) Quero, G. M.; Fusco, V.; Cocconcelli, P. S.; Owczarek, L.; Borcakli, M.; Fontana, C.; Skapska, S.; Jasinska, U. T.; Ozturk, T.; Morea, M. Microbiological, physico-chemical, nutritional and sensory characterization of traditional Matsoni: Selection and use of autochthonous multiple strain cultures to extend its shelf-life. Food Microbiol. 2014, 38, 179−191. (10) Kiryu, T.; Kiso, T.; Nakano, H.; Ooe, K.; Kimura, T.; Murakami, H. Involvement of Acetobacter orientalis in the production of lactobionic acid in Caucasian yogurt (“Caspian Sea yogurt”) in Japan. J. Dairy Sci. 2009, 92, 25−34. (11) Uchida, K.; Akashi, K.; Motoshima, H.; Urashima, T.; Arai, I.; Saito, T. Microbiota analysis of Caspian Sea yogurt, a ropy fermented milk circulated in Japan. Anim. Sci. J. 2009, 80, 187−192. (12) Simova, E.; Beshkova, D.; Angelov, A.; Hristozova, Ts.; Frengova, G.; Spasov, Z. Lactic acid bacteria and yeasts in kefir grains and kefir made from them. J. Ind. Microbiol. Biotechnol. 2002, 28, 1−6. (13) Garrote, G. L.; Abraham, A. G.; De Antoni, G. L. Characteristics of kefir prepared with different grain:milk ratios. J. Dairy Res. 1998, 65, 149−154. (14) Witthuhn, R. C.; Schoeman, T.; Britz, T. J. Characterisation of the microbial population at different stages of Kefir production and Kefir grain mass cultivation. Int. Dairy J. 2005, 15, 383−389. (15) Nielsen, M. S.; Martinussen, T.; Flambard, B.; Sørensen, K. I.; Otte, J. Peptide profiles and angiotensin-I-converting enzyme inhibitory activity of fermented milk products: Effect of bacterial strain, fermentation pH, and storage time. Int. Dairy J. 2009, 19, 155− 165. (16) Delgado, F. J.; González-Crespo, J.; Cava, R.; García-Parra, J.; Ramírez, R. Characterisation by SPME−GC−MS of the volatile profile of a Spanish soft cheese PDO Torta del Casar during ripening. Food Chem. 2010, 118, 182−189. (17) Miyakawa, T.; Liang, T.; Tanokura, M. NMR-based metabolomics of foods. In Genomics, Proteomics and Metabolomics in Nutraceuticals and Functional Foods; Bagchi, D., Swaroop, A., Bagchi, M., Eds.; John Wiley & Sons, Ltd.: Chichester, U.K., 2015; Chapter 29, pp 379−387, DOI: 10.1002/9781118930458.ch29. (18) Wei, F.; Furihata, K.; Miyakawa, T.; Tanokura, M. A pilot study of NMR-based sensory prediction of roasted coffee bean extracts. Food Chem. 2014, 152, 363−369. (19) Wei, F.; Furihata, K.; Hu, F.; Miyakawa, T.; Tanokura, M. Twodimensional 1H-13C nuclear magnetic resonance (NMR)-based comprehensive analysis of roasted coffee bean extract. J. Agric. Food Chem. 2011, 59, 9065−9073. (20) Wei, F.; Furihata, K.; Koda, M.; Hu, F.; Kato, R.; Miyakawa, T.; Tanokura, M. 13C NMR-based metabolomics for the classification of green coffee beans according to variety and origin. J. Agric. Food Chem. 2012, 60, 10118−10125. (21) Agiomyrgianaki, A.; Petrakis, P. V.; Dais, P. Influence of harvest year, cultivar and geographical origin on Greek extra virgin olive oils composition: A study by NMR spectroscopy and biometric analysis. Food Chem. 2012, 135, 2561−2568. (22) Koda, M.; Furihata, K.; Wei, F.; Miyakawa, T.; Tanokura, M. Metabolic discrimination of mango juice from various cultivars by band-selective NMR spectroscopy. J. Agric. Food Chem. 2012, 60, 1158−1166. (23) Schievano, E.; Peggion, E.; Mammi, S. 1H nuclear magnetic resonance spectra of chloroform extracts of honey for chemometric determination of its botanical origin. J. Agric. Food Chem. 2010, 58, 57−65. (24) Koda, M.; Furihata, K.; Wei, F.; Miyakawa, T.; Tanokura, M. NMR-based metabolic profiling of rice wines by F2-selective total correlation spectra. J. Agric. Food Chem. 2012, 60, 4818−4825.

method. Furthermore, the in situ quantitative NMR method for eliminating interference as a result of overlapping signals was shown to be a promising method that could be used to study microorganisms and in the real-time monitoring of chemical changes during fermentation. This would help estimate microbial metabolism in various flora and guide the fermentation processes and storage of various fermented milk products to improve their quality, which can directly influence human health.

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.jafc.7b05279. Assignments of 1H and 13C signals of compounds in Bulgarian yogurt (pH 5.4) (Table S1), assignments of 1H and 13C signals of compounds in Caspian Sea yogurt (pH 4.8) (Table S2), assignments of 1H and 13C signals of compounds in kefir (pH 5.8) (Table S3), 1H NMR spectra of the three fermented milk products that were prepared in different scales (Figure S1), quantitative 1H NMR, DOSY, and the difference spectra of Bulgarian yogurt (Figure S2), quantitative 1H NMR, DOSY, and the difference spectra of Caspian Sea yogurt (Figure S3), and quantitative 1H NMR, DOSY, and the difference spectra of kefir (Figure S4) (PDF)

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AUTHOR INFORMATION

Corresponding Author

*Telephone: +81-3-5841-5165. Fax: +81-3-5841-8023. E-mail: [email protected]. ORCID

Masaru Tanokura: 0000-0001-5072-2480 Funding

This work was supported by a Grant-in-Aid for Scientific Research (S) from the Japan Society for the Promotion of Science (JSPS) (Grant 23228003). Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS The authors thank K. Furihata and F. Wei for assisting with the NMR measurements. REFERENCES

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