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Real-Time Monitoring of Chemical Changes in Three Kinds of Fermented Milk Products During Fermentation Using Quantitative Difference NMR Spectroscopy Yi Lu, Hiroto Ishikawa, Yeondae Kwon, Fangyu Hu, Takuya Miyakawa, and Masaru Tanokura J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.7b05279 • Publication Date (Web): 17 Jan 2018 Downloaded from http://pubs.acs.org on January 22, 2018

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Journal of Agricultural and Food Chemistry

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Real-Time Monitoring of Chemical Changes in Three Kinds of

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Fermented Milk Products During Fermentation Using Quantitative

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Difference NMR Spectroscopy

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Yi Lu, Hiroto Ishikawa, Yeondae Kwon, Fangyu Hu, Takuya Miyakawa, Masaru

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Tanokura*

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Department of Applied Biological Chemistry, Graduate School of Agricultural and Life

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Sciences, The University of Tokyo, 1-1-1 Yayoi, Bunkyo-ku, Tokyo 113-8657, Japan.

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*Corresponding author (Tel: +81-3-5841-5165; Fax: +81-3-5841-8023; E-mail:

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[email protected])

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ACS Paragon Plus Environment


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ABSTRACT: Fermented milk products are rising in popularity throughout the world

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because of their health benefits, including improving digestion, normalizing the function

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of the immune system, and aiding in weight management. This study applies an in situ

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quantitative NMR method to monitor chemical changes in three kinds of fermented

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milk products, Bulgarian yogurt, Caspian Sea yogurt and kefir, during fermentation. As

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a result, the concentration changes in nine organic compounds, α/β-lactose,

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α/β-galactose, lactic acid, citrate, ethanol, lecithin, and creatine, were monitored in real

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time. This revealed three distinct metabolic processes in the three fermented milk

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products. Moreover, pH changes were also determined by variations in the chemical

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shift of citric acid during the fermentation processes. These results can be applied to

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estimate microbial metabolism in various flora and help guide the fermentation and

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storage of various fermented milk products to improve their quality, which may directly

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influence human health.

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KEYWORDS: fermented milk products, in situ quantitative monitoring, NMR

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INTRODUCTION

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Potential health benefits have become key factors for consumers when making their

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food choices.1 Fermented milk products, which are a functional dairy food, have risen in

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popularity throughout the world because several lines of evidence have shown that

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fermented milk products exhibit many health benefits.2-4 The epidemiologic study also

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showed that common effects of fermented milks on an intestinal environment are

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increases in the counts of bifidobacteria and decreases in the counts of harmful

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bacteria.3 These effects may increase during fermentation,5 so it is important that the

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fermentation processes are monitored and controlled. Among the various kinds of

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fermented milk products, Bulgarian yogurt, Caspian Sea yogurt and kefir are widely

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consumed as health foods and for their sensory properties.4,6,7

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Bulgarian yogurt is the most popular variety of yogurt in the world. It was invented

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in Bulgaria and is part of a heritage that dates back many centuries.6 Bulgarian yogurt is

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commonly made with two starter bacteria, Lactobacillus bulgaricus and Streptococcus

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thermophiles, which give the yogurt its characteristic thickness, acidity, taste and

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aroma.8 S. thermophiles bacteria act first and prepare the proper environment for L.

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bulgaricus, which can begin to multiply and slowly turn the milk into yogurt.2 Caspian

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Sea yogurt, also known as Matsoni yogurt, is one of the yogurts that is cultured at room

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temperature.9 It originates from the Caucasus region between the Black Sea and Caspian

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Sea, a region famous for the longevity of the population.10 The microbiota of Caspian

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Sea yogurt is reported to be Lactococcus lactis subsp. cremoris (Streptococcus

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cremoris) and Acetobacter orientalis, which give it its uniquely viscous consistency.11

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Kefir is a fermented milk product made with kefir grains, consists of lactic acid bacteria

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and yeasts and is obtained by a combined acidic and alcoholic fermentation.12 It

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originates from the Caucasian mountains.13 The microbial populations in the kefir grain

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are Lactobacillus, Lactococcus and yeast.14

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Recently, studies on chemical compositions of fermented milk products have been

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carried out.15,16 In these studies, organic acids, acetaldehyde and other compounds in

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fermented milk were extracted, separated, and then analyzed by LC-MS15 and

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GC-MS.16 In all these steps (extraction, separation and derivatization), even simple

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treatments could cause qualitative and quantitative changes to the original mixture.

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NMR is a highly quantitative and reproducible analytical technique.17 As a non-targeted

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method, NMR measurements do not require any separation or chemical modification,

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giving overall information regarding the chemical components of a mixture rapidly and

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directly. Therefore, NMR spectroscopy would allow us to perform in situ monitoring

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and quantitative analysis in real time. In the last few decades, NMR has been recognized

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as a powerful technique that has been widely used for metabolic description of foods,

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such as coffee,18-20 olive oil,21 juice,22 honey,23 and liquor as a fermented food.24,25 In

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addition, milk has been analyzed by NMR to identify and quantitate characteristic

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compounds,26,27 and overview metabolite patterns for quality assessments.28-31

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More recently, an in situ quantitative method using difference spectra between

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quantitative 1H NMR spectra and diffusion ordered spectroscopy (DOSY) NMR spectra

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were developed for measuring the components in fermented milk products.32 In the

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present study, we applied the in situ quantitative NMR method to monitor the

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fermentation processes of different kinds of fermented milk products in real time. The

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obtained quantitative data could be used to guide the fermentation processes and storage

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of various fermented milk products, improving the quality of the fermented milk

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products which can directly influence human health.

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MATERIALS AND METHODS

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Materials and Sample Preparation. The inoculums of Bulgarian yogurt

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(Lactobacillus bulgaricus, Streptococcus thermophilus and skim milk) (AFC Co., Ltd.,

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Shizuoka, Japan) and Caspian Sea yogurt (Streptococcus cremoris FC, Acetobacter

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orientalis FA and skim milk) (Fujicco Co., Ltd., Kobe, Japan), kefir grains

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(Lactobacillus casei strain Shirota, Streptococcus cremoris, Saccharomyces florentinus

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and skim milk) (Nihon kefir Co., Ltd., Fujisawa, Japan) and whole milk (Meiji Co., Ltd.,

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Tokyo, Japan) were purchased at a local market. For fermentation monitoring, the

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inoculums of Bulgarian yogurt (1 g), Caspian Sea yogurt (6 g) and kefir grains (4 g)

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were each added to whole milk (1 L). The samples were immediately mixed with D2O

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to give final concentrations of 10% (v/v) and were then placed in 5 mm NMR tubes.

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The volumes of the samples were adjusted to 0.6 mL. A capillary containing 20% (v/v)

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1,1,2,2-tetrachloroethane (Wako Pure Chemical Co. Ltd., Osaka, Japan), 80% (v/v)

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chloroform-d (Isotec Inc., Tokyo, Japan) and 1 mg/mL chromium(III) acetylacetonate

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(Kanto Chemical Co. Ltd., Tokyo, Japan) was inserted into each NMR tube as the

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concentration standard.32 Each inoculum sample was prepared in triplicate from the

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same fermentation batch.

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NMR Spectroscopy. NMR measurements were performed at 40 °C (Bulgarian

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yogurt) or 25 °C (Caspian Sea yogurt and kefir) on a Unity INOVA-500 spectrometer

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(Agilent Technologies, Santa Clara, CA) for the 1H quantitative NMR spectra (time for

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data collection, 10 min) and diffusion ordered spectroscopy (DOSY) NMR spectra

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(time for data collection, 5 min). The prepared samples were placed in the NMR

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equipment and the first sequential acquisition of the 1H quantitative and DOSY NMR

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spectra was carried out within 15 min. The collected spectra were defined as time 0. The

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fermentation was then performed on the NMR equipment at 40 °C for 32 h (Bulgarian

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yogurt), 25 °C for 22 h (Caspian Sea yogurt) and 25 °C for 25 h (kefir) without spinning

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or stirring. The 1H NMR spectra were acquired at 5, 10, 12, 14, 16, 18, 20, 22, 24, 26,

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28, 30 and 32 h for Bulgarian yogurt; 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 20 and 22 h

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for Caspian Sea yogurt; and 11, 13, 15, 17, 19, 21, 23, 24 and 25 h for kefir during

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fermentation. At the endpoint, each sample was smelled and tasted similarly to the

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products prepared according to manufactures’ standard protocols.

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The 1H NMR spectra of the fermented milk products were measured at 499.87

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MHz, and an HDO signal was suppressed by pre-saturation. The CH-β2 signal of

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lactose was used as an internal reference, and its chemical shift was set to 3.16 ppm

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based on the data of our previous study.32 For quantitation, the acquisition parameters

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were as follows: number of data points, 32 K; spectral width, 8,000 Hz; acquisition time,

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2.048 s; delay time, 15 s; and number of scans, 32. The delay time (d1) was determined

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with the spin-lattice relaxation time (T1) and the acquisition time (aq). ≥ 5 × − aq

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The DOSY NMR measurements were carried out soon after each series of 1H NMR

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measurements was complete. The acquisition parameters were as follows: number of

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data points, 32 K; spectral width, 8000 Hz; acquisition time, 2.048 s; delay time, 15 s;

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number of scans, 16; diffusion delay, 0.4 s; total diffusion-encoding gradient pulse

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duration, 0.002 s; and gradient stabilization delay, 0.0003 s. The signals that overlapped

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with the signals of milk fats were quantitated using difference spectra that were

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obtained by subtracting the DOSY spectra from the quantitative 1H NMR spectra.32

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For NMR signal assignments,

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C and 2D NMR spectra (1H-13C HSQC, 1H-1H

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DQF–COSY, and 1H-13C CT-HMBC) of the three fermented milk products (32-h

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fermentation at 40 °C for Bulgarian yogurt, 22-h fermentation at 25 °C for Caspian Sea

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yogurt, and 25-h fermentation at 25 °C for kefir) were measured at 4 °C on a Unity

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INOVA-500 spectrometer.

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The

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C NMR spectra were measured at 125.71 MHz. Dioxane was used as an

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external reference, and its chemical shift was set to 67.5 ppm. The parameters of the 13C

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NMR spectrum were as follows: number of data points, 64 K; spectral width, 31,422

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Hz; acquisition time, 1.043 s; delay time, 2 s; and number of scans, 83,392.

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The 1H-1H DQF-COSY spectra were obtained by suppressing the water signal with

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pre-saturation, and the acquisition parameters were as follows: number of data points,

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2048 (F2) and 512 (F1); spectral width, 5911 Hz (F1 and F2); acquisition time, 0.202 s;

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delay time, 2 s; and number of scans, 48.

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The

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H-13C HSQC spectra of the fermented milk were generated in the

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phase-sensitive mode with the following acquisition parameters: number of data points,

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512 for 1H and 256 for

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acquisition time, 0.186 s; delay time, 2 s; and number of scans, 80.

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C; spectral widths, 5498 Hz for 1H and 20,110 Hz for

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C;

The 1H-13C CT-HMBC spectra were measured in the absolute mode with the

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following parameters: number of data points, 4096 for 1H and 512 for

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widths, 5498 Hz for 1H and 27,643 Hz for 13C; acquisition time, 0.402 s; delay time, 3 s;

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and number of scans, 80.

C; spectral

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NMR Signal Assignments and Data Processing. The preprocessing of the

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free-induction decays (FIDs) and the subsequent Fourier transformations were

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performed by the program MestRe Nova 10.0 (MestRec, Santiago de Compostela,

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Spain). NMR signals were analyzed by comparison to our previous published data

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based on two-dimensional NMR correlations, including NMR assignment data32 and

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composition data.33,34 The signals were then confirmed and assigned to the candidate

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compounds using the 2D NMR spectra. The quantitative method developed in our

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previous study32 was used to investigate the changes in the chemical composition over

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time. The integral value of 1,1,2,2-tetrachloroethane was compared with those of the

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compounds in the three fermented milk products to determine their concentrations.

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pH Measurements. Citrate-phosphate buffer (0.1 M) prepared with D2O was

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adjusted to each pH value (pH 4.0‒6.8) using a Twin pH B-212 pH meter (Horiba,

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Kyoto, Japan). The 1H NMR spectrum of each buffer was recorded at 25 °C on a Unity

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INOVA-500 spectrometer. The standard pH titration curve was created by plotting

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chemical shifts of citric acid (CH-β2) against the pH values. The pH values during the

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fermentation process were calculated using the standard pH titration curve and the

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chemical shift values of citric acid (CH-β2).

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RESULTS AND DISCUSSION

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NMR Spectroscopic Analysis of the Three Fermented Milk Products. NMR

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spectral analyses of the three fermented milk products were carried out to monitor the

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chemical changes during fermentation on a solution-state NMR spectrometer. Figure 1

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shows quantitative 1H NMR spectra of the three fermented milk products during

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fermentation. The initial sample (0 h) looked like a solution because of the mixture of

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milk and a small amount of each inoculum. On the other hand, the final samples (32, 25

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and 22 h for Bulgarian yogurt, Caspian Sea yogurt and kefir, respectively) were

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semi-solid. Although the states are different between initial and final samples, the

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observed spectra, especially the signal shapes, were quite similar. In addition, the 1H

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NMR spectra of the final samples were quite similar to those of the samples of a

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100-mL scale-up fermentation. Based on 2D NMR analysis, spiking experiments and

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peak assignments reported in a previous study,32 nine components were identified:

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α/β-lactose, α/β-galactose, lactic acid, citrate, lecithin, creatine and ethanol. As shown in

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Figure 1B, signals indicative of galactose were not observed during fermentation of

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Caspian Sea yogurt, unlike Bulgarian yogurt and kefir in which galactose was observed.

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As shown in Figure 1C, ethanol was only observed in kefir.

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Changes in Component Concentrations in the Three Fermented Milk Products.

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Our developed quantitation method32 was used to quantitate the concentrations of

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components in the three fermented milk products during fermentation. The integral

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values of signals were calculated on the difference spectra that were obtained by

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subtracting the DOSY spectra from the quantitative 1H NMR spectra. The 1H signal

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(5.83 ppm) of 1,1,2,2-tetrachloroethane was used as an external quantitative standard

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because it was separately observed from other signals. The nine detected compounds in

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the three fermented milk products were quantitated by comparing the integral values of

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their signals with that of the standard signal. The quantitative results are shown in

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Figures 2, 3 and 4. Overall, the patterns of concentration changes were different among

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the three fermented milk products even when the same compound was compared,

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especially citric acid and α/β-galactose. The concentration of α/β-lactose and citric acid

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were 146‒185 mmol/L (50‒63 g/L) and 3.1‒4.5 mmol/L in the initial sample (0 h),

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respectively. These values were not largely different from those in milk that was

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determined by a previous NMR study (47.8 g/L for α/β-lactose and 3.2 mmol/L for

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citric acid).27 The concentration of the nine detected compounds was hardly affected, by

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comparison of the initial sample (0 h) with the sample after fermentation for about 10 h.

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Chemical Changes in Bulgarian Yogurt During the Fermentation Process. As

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shown in Figure 2, the quantities of lactic acid and citric acid did not vary greatly during

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the first 12 h or during the last 8 h, but increased quickly between 12 and 24 h of

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fermentation. Continuous decreases in α/β-lactose were detected starting at 14 h, while

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the concentrations of α/β-galactose increased from 12 to 32 h. No obvious changes in

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the concentrations of lecithin or creatine were observed during fermentation.

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The α/β-lactose ratio observed during the fermentation process was 1:1.57 ± 0.014 (data

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is mean ± standard deviation), which is slightly higher than that of the water solution

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(1:1.5 at 25 °C). The temperature for NMR measurements (40 °C) and the composition

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of

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α/β-lactose ratio.35 The ratio was not largely changed during fermentation.

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organic

compounds

in

Bulgarian

yogurt

products

may

influence

the

L. bulgaricus and S. thermophiles are classified into lactic acid bacteria that

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generally decompose lactose to glucose and galactose and produce lactic acid by the

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glycolytic pathway and lactic acid fermentation (Figure 5). As shown in Figure 2, the

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increased production of lactic acid was observed at 14 h, which corresponds to the

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starting time of α/β-lactose consumption. Interestingly, the 1H NMR signals of glucose

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were not observed in the observed fermentation period (Figure 1A), which suggests that

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glucose from lactose degradation is immediately consumed in the glycolytic pathway.

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On the other hand, α/β-galactose began to increase at 12 h of fermentation. Furthermore,

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the increased amount of galactose (7.7 ± 0.7 mmol/L) during fermentation from 10 h to

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12 h was much higher than the amount of lactose consumption (2.2 ± 0.4 mmol/L)

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(Figure 2). This observation raises a possibility that galactose may be produced from

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multiple pathways, e.g. degradation of galactooligosaccharides,36 in addition to the

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hydrolysis of lactose. Citric acid was observed in the initial sample (0 h) in agreement

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with the citric acid content of milk. It is well known that citric acid is a constituent of

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the tricarboxylic acid (TCA) cycle and is produced from glucose through the glycolytic

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pathway and the oxidative conversion of pyruvic acid into acetyl-CoA that is carried out

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by pyruvate dehydrogenase (Figure 5).37,38 The rapid increase in the amount of citric

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acid is thought to be due to the mass production of pyruvic acid between 16 and 24 h

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(Figure 2), which does not contradict with the result of lactic acid that is also produced

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by pyruvate during lactic acid fermentation. However, the concentration of citric acid

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started to be elevated at 16 h, which is later than the starting point of lactic acid

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production. In addition, the production rate of lactic acid was three-fold higher than that

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of citric acid. These observations suggest that the pyruvic acid could be mainly used in

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the lactic acid fermentation.

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Lecithin is one of the major components of the phospholipid portion of the cell

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membrane39 and is a dietary source of choline, which can be converted into

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acetylcholine, an organic compound that functions as a neuromodulator in the brain.40

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Creatine can be phosphorylated to form phosphocreatine, which is recognized as a kind

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of energy buffer in skeletal muscles and in the brain.41 These nutritional compounds

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were hardly affected during the fermentation of Bulgarian yogurt.

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Chemical Changes in Caspian Sea Yogurt During the Fermentation Process.

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As shown in Figure 3, the amount of lactic acid did not vary substantially during the

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first 12 h or the last 7 h, but increased rapidly between 12 and 15 h of fermentation. The

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concentration of citric acid increased quickly between 12 and 14 h and then decreased

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sharply between 14 and 18 h of fermentation. Continuous decreases in α/β-lactose were

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observed between 9 and 20 h. The quantities of lecithin and creatine did not vary

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substantially during fermentation. The α/β-lactose ratio observed in the present study

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was 1:1.64 ± 0.018 (mean ± standard deviation), which is slightly higher than that of the

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water solution (1:1.5 at 25 °C). The ratio was not largely changed during fermentation.

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Unlike in Bulgarian yogurt, signals from galactose were not observed in Caspian

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Sea yogurt during fermentation. Galactose is incorporated into the cells by

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phosphoenolpyruvate: carbohydrate phosphotransferase system of S. cremoris.42 The

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system could be one of the reasons why the consumption of galactose during the

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fermentation of Caspian Sea yogurt was much faster than that in Bulgarian yogurt.

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Caspian Sea yogurt produces exopolysaccharides (EPS) during fermentation to become

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more viscous,11 and since galactose is a building block of EPS, it may be consumed and

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used in EPS production during the fermentation of Caspian Sea yogurt (Figure 5).43 The

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increase in citric acid between 12 and 14 h is likely caused by the mass production of

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pyruvate during lactic acid fermentation, whereas citric acid was decreased between 14

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and 18 h. Lactic acid bacteria are also able to convert citric acid to some end products,

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such as diacetyl and acetaldehyde, which contribute to the quality of the fermented

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foods due to their distinct aroma properties.44,45 The metabolic flow of citric acid seems

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to be converted from production to degradation when lactic acid fermentation was

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terminated at 15 h. This metabolic change was not observed in Bulgarian yogurt, which

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may be affected by differences in microbial compositions between Bulgarian yogurt and

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Caspian Sea yogurt.

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Chemical Changes in Kefir During the Fermentation Process. As shown in

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Figure 4, the concentration of lactic acid increased slowly between 11 and 17 h, and

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from 23 to 25 h during fermentation and increased quickly between 19 and 23 h. Citric

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acid decreased sharply between 11 and 25 h, and eventually disappeared. Lactose and

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galactose decreased substantially between 11 and 25 h of fermentation. The signals of

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ethanol were observed at 17 h, and the concentration of ethanol increased between 15

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and 25 h of fermentation. The amount of lecithin and creatine did not vary to a large

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extent during fermentation. The α/β-lactose ratio observed in the present study was

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1:1.55 ± 0.018 (data is mean ± standard deviation), which is slightly higher than that of

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the water solution (1:1.5 at 25 °C) and is not largely changed during fermentation.

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Kefir grain mainly contains Lactobacillus casei, S. cremoris and Saccharomyces

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florentinus in addition to a mixture of proteins, lipids, and sugars. Lactose could be

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decomposed to produce glucose and galactose by lactic acid bacteria as well as in

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Bulgarian yogurt and Caspian Sea yogurt. The metabolic pathway of yeast (Figure 5),46

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which includes an alcohol fermentation pathway, is different from that of lactic acid

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bacteria. Yeast begins to work after the pH becomes sufficiently low, eventually

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producing ethanol by consumption of glucose. The quantitative results show that the

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similar chemical changes of lactic acid and lactose observed in Bulgaria yogurt and

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kefir were due to the action of Lactobacillus and Streptococcus, while ethanol was

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produced by yeast. This is the characteristic of kefir that makes it different from the

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other two kinds of yogurt. The glucose fermentation of yeast was monitored by NMR

284

spectroscopy.47 As the reaction proceeds, the glucose signal was decreased while the

285

intensity of ethanol signals was increased, which corresponds to the results of kefir

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fermentation in the present study. Citric acid level decreased during the fermentation of

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kefir by the citric acid metabolism. The consumption rate of citric acid was the fastest

288

among three fermented milk products in this study. On the other hand, production of

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citric acid in kefir may be less than those in other fermented milk products because

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pyruvate is also consumed by ethanol fermentation in addition to lactic acid

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fermentation (Figure 5). Kefir has unique and complex probiotic properties in which

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kefiran often functions as a promising compound. Kefiran is known to be a

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water-soluble EPS produced in kefir grain during fermentation that has anticancer,

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anti-inflammatory, and hypocholesterolenic effects, and can aid in managing the

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immune system and lowering blood pressure.48 Decreased galactose levels during kefir

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fermentation (Figure 4) suggest the production of kefiran because it is a galactose-rich

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polysaccharide.

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Relative Changes in Fatty Acids During the Fermentation Processes. To

299

investigate the changes in the composition of fatty acids during the fermentation

300

processes, the step-by-step integral values of the signals at 0.76 ppm were calculated

301

(Figure 6). Fatty acids are naturally found in dairy products, and trans fatty acids are

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associated with biological effects such as increased risk of coronary heart disease,

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which may directly affect human health.49 Furthermore, fatty acid synthesis, which

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occurs in the cytoplasm of the cell, is the process by which fatty acids are produced

305

from acetyl-CoA and NADPH by enzymes.50 In this study, integrals of fatty acids

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signals did not change substantially during the fermentation processes, which indicated

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that the concentrations of NMR-detected fatty acids did not vary very much during

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fermentation.

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pH Changes in the Three Fermented Milk Products During Fermentation.

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Figure 7A shows the relationship between chemical shift of citric acid and pH of a

311

citrate-phosphate buffer. Figure 7B shows the changes in the 1H chemical shift of citrate

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in the three fermented milk products during fermentation. The pH changes in Caspian

313

Sea yogurt during fermentation were observed by two different methods. The pH values

314

were first calculated by the chemical shifts of citric acid signals in the 1H NMR spectra

315

during fermentation (Figures 7A and B). Then, the pH values calculated from the NMR

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spectra were compared with the values determined by a pH meter (shown in Figure 7C).

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These results revealed that the pH values calculated during the fermentation of Caspian

318

Sea yogurt were consistent with those measured by the pH meter. The present study is

319

the first application of the pH measurements from NMR spectra during fermentation of

320

yogurt. The pH levels during the fermentation of Bulgarian yogurt and kefir were also

321

calculated from NMR spectra (Figure 7C). This method provides a real-time,

322

non-destructive way to confirm pH during fermentation of yogurt, which is required for

323

quality control of yogurt.

324

In conclusion, this study applied our newly developed quantitative NMR method

325

for monitoring chemical changes in various fermented milk products during

326

fermentation. The acquisition of quantitative 1H NMR spectra and DOSY NMR spectra

327

are easy and quick, and the preparation of samples in organic solvent is simple, both of

328

which are important advantages to this method. Furthermore, the in situ quantitative

329

NMR method for eliminating interference due to overlapping signals was shown to be a

330

promising method that could be used to study microorganisms and in the real-time

331

monitoring of chemical changes during fermentation. This would help estimate

332

microbial metabolism in various flora and guide the fermentation processes and storage

333

of various fermented milk products to improve their quality which can directly

19



334

influence human health.

335

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ASSOCIATED CONTENT

337

Supporting Information

338

The Supporting Information is available free of charge on the ACS Publications

339

website.

340

Assignments of 1H and

13

341

assignments of 1H and

C signals of compounds in Caspian Sea yogurt (pH 4.8);

342

assignments of 1H and 13C signals of compounds in kefir (pH 5.8); 1H NMR spectra of

343

the three fermented milk products that were prepared in different scale; quantitative 1H

344

NMR, DOSY, and the difference spectra of Bulgarian yogurt; quantitative 1H NMR,

345

DOSY, and the difference spectra of Caspian Sea yogurt; and quantitative 1H NMR,

346

DOSY, and the difference spectra of kefir (PDF)

C signals of compounds in Bulgarian yogurt (pH 5.4);

13

347

348

AUTHOR INFORMATION

349

Corresponding Author

350

*Tel:

351

[email protected]

352

ORCID

353

Masaru Tanokura: 0000-0001-5072-2480

+81-3-5841-5165;

Fax:

+81-3-5841-8023;

21


E-mail:


354

Funding

355

This work was supported by a Grant-in-Aid for Scientific Research (S) from the Japan

356

Society for the Promotion of Science (JSPS) (Grant No. 23228003).

357

Notes

358

The authors declare no competing financial interest.

359

360

ACKNOWLEDGMENTS

361

We thank K. Furihata and F. Wei for assisting with the NMR measurements.

362

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515

FIGURE CAPTIONS

516

Figure 1. Quantitative 1H NMR spectra of (A) Bulgarian yogurt, (B) Caspian Sea

517

yogurt, and (C) kefir during fermentation. HDO signals (4.4–4.8 ppm) were suppressed.

518

519

Figure 2. Evolution of components in Bulgarian yogurt during fermentation. Data are

520

means ± standard deviations (SD, n = 3).

521

522

Figure 3. Evolution of components in Caspian Sea yogurt during fermentation. Data are

523

means ± standard deviations (SD, n = 3).

524

525

Figure 4. Evolution of components in kefir during fermentation. Data are means ±

526

standard deviations (SD, n = 3).

527

528

Figure 5. Schematic diagram of major metabolic pathways during the fermentation of

529

Bulgarian yogurt (pink arrows), Caspian Sea yogurt (cyan arrows) and kefir (orange

530

arrows). The thickness of arrow represents the pseudo-rate of metabolic flow. The

531

bacterial strains most common in Bulgarian yogurt are Lactobacillus bulgaricus and

532

Streptococcus thermophiles. Caspian Sea yogurt is composed of Streptococcus cremoris

32


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533

and Acetobacter orientalis. The microbial populations in the kefir grain are

534

Lactobacillus casei, S. cremoris and Saccharomyces florentinus. EPS means

535

exopolysaccharide.

536

537

Figure 6. Relative changes in fatty acid levels during the fermentation of the three

538

fermented milk products. Data are means ± standard deviations (SD, n = 3).

539

540

Figure 7. (A) The relationship between the chemical shift of citric acid and pH of a

541

citrate-phosphate buffer (25 °C). The CH-β2 (3.16 ppm) signal of lactose was used as an

542

internal reference. (B) 1H chemical shift changes of citrate in the three fermented milk

543

products during fermentation. (C) pH changes in the three fermented milk products

544

during fermentation. The blue line represents the chemical shifts of citric acid and the

545

pH values calculated from the NMR spectra during the fermentation of Caspian Sea

546

yogurt. The purple line represents the pH values measured by a pH meter during the

547

fermentation of Caspian Sea yogurt. The red line represents the chemical shifts of citric

548

acid and the pH values calculated from the NMR spectra during the fermentation of

549

Bulgarian yogurt. The light green line represents the chemical shifts of citric acid and

550

the pH values calculated from the NMR spectra during the fermentation of kefir.

33



Figure 1. Lu et al. 34


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Figure 2. Lu et al.

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Figure 3. Lu et al.

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Figure 5. Lu et al.

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Figure 6. Lu et al.

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Figure 7. Lu et al.

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Real-Time Monitoring of Chemical Changes in Three Kinds of

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