JOURNAL OF FERMENTATION AND BIOENGINEEPWG

Vol. 84, No. 5, 461465.

1997

Prediction of the Concentrations of Ethanol and Acetic Acid in the Culture Broth of a Rice Vinegar Fermentation Using Near-Infrared Spectroscopy
TAKUO YANO,* TADANORI AIMI, YASUHISA NAKANO,
AND

MASAHIRO

TAMAF

Department

of Information Machines and Interfaces, Faculty of Information Sciences, Hiroshima City University, 3-4-l Ohzukahigashi, Asaminami-ku, Hiroshima 731-31 and Hiroshima Prefectural Food Technology Research Center, 12-70 Hijiyamahonmachi, Minami-ku, Hiroshima 732,2 Japan
Received 20 November 1996/Accepted 4 August 1997

Near-infrared spectroscopy (NIR), which is a nondestructive analytical technique, was employed for the simultaneous prediction of the concentrations of ethanol and acetic acid in the culture broth sampled from a rice vinegar fermentation. The broth was placed in a near-infrared spectrophotometer and the absorbance at wavelengths between 400 and 2,SOOnm was measured at 2nm intervals. To obtain calibration equations, multiple linear regression (MLR) was conducted on the NIR spectral data and on the ethanol and acetic acid concentrations obtained by gas chromatography of a calibration sample set. The value of the multiple correlation coefficient (R) was 0.999 when using the wavelengths of 1,686 and 1,738 nm for ethanol. The value of R for acetic acid was 0.940 when using the wavelengths of 1,674 and 1,718 nm. To validate the calibration equations obtained, ethanol and acetic acid concentrations in a prediction sample set which was not used for calibration were calculated using the calibration equation, and compared with the concentrations measured by gas chromatography. Excellent agreement between the results of the conventional method and those of NIR was observed for both constituents. The concentrations of ethanol and acetic acid in the culture broth could be analyzed simultaneously by NIR. The procedure of NIR was simple, and the operation time required to predict the concentrations was only 5 min. These results indicate that NIR may be a useful method for the monitoring and control of rice vinegar fermentation. [Key words: rice vinegar fermentation, near-infrared spectroscopy]

In the cultivation of organisms, it is very important, for the management of the process, to monitor the concentrations of substrates and products. In vinegar fermentation, gas chromatoghraphy is used to measure the concentrations of ethanol and acetic acid in the culture broth. Organic acids such as gluconic and 2-ketoglutaric acids, which affect the taste of the vinegar, are measured by liquid chromatography, while, the cell density of the culture broth is calculated on the basis of the optical density measured using a spectrophotometer. The development of a simple method of measuring the concentrations of these constituents simultaneously has been desired for a long time. In the present study, to determine the potential of near-infrared spectroscopy (NIR) for use in the management of the rice vinegar fermentation process, NIR was used to predict the concentrations of ethanol and acetic acid in the culture broth sampled from several runs of the fermentation. We discuss the assignment of the wavelength selected for the prediction. Optical density of the culture broth was also measured using a nearinfrared spectrophotometer. NIR is widely used for rapid and nondestructive analysis in industries, such as agriculture, food, pharmaceuticals, textiles, cosmetics and polymer production industries (1). In fermentation processes, NIR has been applied to measure the ethanol concentrations in fermentations with molasses (2) and wine (3) and beer (4, 5). The concentrations of glucose, glutamine, ammonia and lactic acid in the supernatant of an animal cell culture have * Corresponding author.
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also been simultaneously

MATERIALS Materials

analyzed
AND

by NIR (6).
METHODS

Samples of the culture broth of a rice vinegar fermentation (number of samples, n=63) were removed from a 23 m3 fermentor (working volume: 17 m3), called the Acetator (Heinrich Frings GmbH, Bonn), set up in a vinegar brewery. The samples were drawn from several runs of the fermentation, and contained ethanol and acetic acid ranging in concentrations from 5.7 to 34.8g/l and from 66.9 to 109g/l, respectively. A set of 42 samples was used as the calibration sample set, and the set of the remaining 21 samples as the prediction sample set. In order to select the wavelength for calibration equations, 40 aqueous samples of an authentic ethanol ranging in concentration from 0 to 78.9 g/l, 60 aqueous samples of mixed authentic ethanol and acetic acid ranging in concentration from 5.6 to 75.9g/l and from 8.9 to 90.6g/l for ethanol and acetic acid, respectively, and 40 aqueous samples of an authentic acetic acid ranging in concentration from 0 to 106.3 g/l were used. Ultrapure reagents of ethanol and acetic acid, commercially supplied by Katayama Chemical Co. (Osaka), were used as authentic reagents. Near-infrared spectroscopy The culture broth was incubated in a water bath to heat it to the required temperature and was then placed in a cuvette with a 2 mm light path length. After putting the cuvette in the cell holder of the near-infrared spectrophotometer (NIRS6500SPL, Nireco, Ishikawa-cho, Hachioji, Tokyo),

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the absorbance from 400 to 2,500nm was measured at 2 nm intervals. The cell holder was maintained at a constant temperature by circulating water from the water bath throughout the jacket of the cell holder. The spectrum of each sample was obtained by taking the average reading of 34 scans. All samples were measured in duplicate and two spectra for each sample were stored on the hard disk (200MB) of a computer (MBC-20CV, Sanyo, Moriguchi, Japan) equipped with Intel-DX-4 CPU (100 MHz), 8MB RAM and MS-DOS 5.0/V. In order to produce calibration equations, NIR spectra of the calibration sample set (n=42) of the culture broth in the rice vineger fermentation were measured. In the mathematical treatment, second derivatives were obtained for the calibration set of spectra as required (7, 8). Multiple linear regression (MLR) using least-squares methods (9) was conducted on the NIR spectral data and on the concentrations obtained by the conventional sample set to obtain methods, C,,,, for the calibration the following calibration equation: Y Cpre=@O+i+,Qi.Ai (1)

/
1000 1200 1400 1600 1600 2000 2200

.I
2400

800

Wavelength

(nm)

FIG. 1. Raw near-infrared spectra of authentic ethanol, authentic acetic acid and the culture broth of a rice vinegar fermentation, and a second-derivative near-infrared spectrum of the culture broth of a rice vinegar fermentation. Lines: authentic ethanol (-), authentic acetic acid (mm-m), raw spectrum (p p) and second-derivative spectrum (- m) of the culture broth.

calculated from where C,,, is the predicted concentration the NIR spectral data, and a is the regression coefficient. The subscript i (i= 1, 2, . . . . . m) represents the number of the wavelength used in the regression analysis. When the calculation is carried out using the raw optical data, A is represented by log(l/T). T is the transmittance value of the sample at each wavelength. When the calculation was carried out using the second-derivative optical data, A at wavelength j (nm) is represented by the following equation and expressed by d210g(l/T): A=Al--2.A2tA3 where Al=[r$llog
(liTj 1.5seg-gap+2k)l/(seg/2+ 1)

1.5 and 1.0 kg/cm2 of pressure, respectively. Temperatures at the injection portion, the column oven and the detector were kept at 210, 180 and 21OC, respectively. The optical density of the culture broth placed in a cuvette with a 10 mm light path length was measured at the wavelength of 660 nm using a spectrophotometer (UV-210, Shimadzu).
-0.95

-0.96
-0.97 c

(2)

I(A)

(3) (4)

-0.98

I
i-

-0.99

..:::
/

s
I I

-1

A3=<;Y;log

(l/T

1+05sep+pap+2k)l/(seg/2+

1)

(5)

800

I1

1000

1200

1400

1600

1800

2000

2200

2400

Wavelength

(nm)
-T

In the equations, seg represents the segment size (nm) used for averaging the values of several data points in the neighborhood of the wavelength, and gap represents the gap size (nm) which is the distance from the nearest data point used in Eq. 4. In this study, 20 and 4nm were used as the values of seg and gap, respectively. To evaluate the performance of the calibration equations obtained, they were validated using the prediction sample set (n=21). The second derivatization, calibration and validation were carried out using the program called NSAS (Near-Infrared Spectral Analysis Software) supplied by Nireco Co. The concentrations of ethaConventional analysis nol and acetic acid in the samples of the culture broth and the authentic compounds were measured using a gas chromatograph (GC-17A, Shimadzu, Kyoto) equipped with a 2 m glass column (2.0 I.D.) packed with Gaskuropack 54 (CL Sciences Co., Tokyo) which is a porous polystyrene-divinylbenzene copolymer. Nitrogen carrier gas was supplied at 1.2 kg/cm* of pressure. Hydrogen and air were supplied to a flame ionization detector at

-0.95

,_II

t (B)
-0.96 t

-1 L800

LL

1000

1200

1400

1600

1800

2000

2200

2400

Wavelength

(nm)

FIG. 2. Plots of correlation coefficient (r) against wavelengrh for ethanol (A) and acetic acid (B) in the calibration sample set. Secondderivative spectra were subjected to simple linear regression. Lines: authentic aqueous solution (~~~~~) and mixture of authentic ethanol and acetic acid (-_).

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RESULTS AND DISCUSSION NIR spectra and second-derivative spectrum In Fig. 1, a raw NIR spectrum and a second-derivative spectrum of the culture broth of the rice vinegar fermentation are plotted between 800 and 2,50Onm, although visible spectra were also measured at the same time. The two main peaks observed at around 1,454 and 1,940 nm on the raw spectrum may be due mainly to the absorption of water (10). Raw NIR spectra of authentic ethanol (99.5% in purity) and acetic acid (99.7%) were measured and are also shown in Fig. 1. The spectrum of authentic ethanol exhibits main peaks at 1,696, 1,732, 2,084, 2,278, 2,310 and 2,354nm. Three main peaks were observed at 1,678, 1,720 and 2,254 nm on the spectrum of authentic acetic acid. Peaks at around 1,700nm may be attributable to the absorption of the carbon-hydrogen (C-H) stretch first overtone (10). The peak at 2,084nm on the ethanol spectrum and at 2,254 nm on the acetic acid spectrum may be caused by a combination of O-H stretching with O-H deformation. On the ethanol spectrum, the peak at 2,278 nm may be caused by a combination of O-H stretching with C-C stretching, and the peaks at 2,310 and 2,354nm may be caused by the C-H group (10). Correlation plots for determining ethanol concentraThe correlations between tion in aqueous samples C,,, for ethanol and the second-derivative values of the
0.05

absorbance of NIR spectra, d210g(1/7), were analyzed using simple linear regression. The values of the correlation coefficient (r) obtained for ethanol are plotted in Fig. 2A. To produce a stable and accurate calibration equation, it is very important to select a wavelength having the absorption assigned to the target compound. Furthermore, a wavelength having a r value close to - 1 should be selected. As shown in Fig. 2A, values of r close to - 1 were observed in 10 wavelength regions in the case of the aqueous samples containing only authentic ethanol. The values of r close to ~ 1 were observed in 5 wavelength regions in the case of the aqueous samples containing authentic ethanol and acetic acid. The absolute values of r decreased to below 0.95 in 5 wavelength regions upon adding acetic acid to ethanol solution. The selection of the wavelength for the ethanol calibration equation was investigated. As shown in Fig. 3A, a peak due to ethanol was observed at 1,688 nm in each spectrum of the aqueous solutions of ethanol. Spectra of aqueous mixed solutions of ethanol and acetic acid are shown in Fig. 3C. In these mixed solutions, ethanol concentration, Cact, was almost the same. In Fig. 3C, it is clear that d210g(1/ZJ at 1,654, 1,686, 1,706 and 1,738 nm is not affected by the concentration of acetic acid. The negative peak observed at 1,688 nm in Fig. 3A was shifted to a shorter wavelength upon adding acetic acid to aqueous ethanol solution. Therefore, the wavelength

0.04 0.03

0.04 0.03

5
-zi 0 w0 -0.01 -0.02

0.02 0.01 0 -0.01

-0.031
1600

1620

1640

1660

1660

1700 (nm)

1720

1740

1760

1600

1620

1640

1660

1660

1700 (nm)

1720

1740

1760

Wavelength

Wavelength
0.05

0.04

c 5 : 0

0.02 0.01 0

li76 -0.02 -0.03 1600 (C)

I,
1620

1
1640

/
1660

I,
1660

,/ 1700 (nm) 1720

,/ 1740 1760

-0.03 1 1600

1620

1640

1660

1660

1700 (nm)

1720

1740

1760

Wavelength

Wavelength

FIG. 3. Second-derivative near-infrared spectra of aqueous samples of authentic ethanol (A), acetic acid (D) and a mixture of authentic ethanol and acetic acid (B, C). Lines: ethanol concentration (g/n, C,,,, in (A) is 72.1 (-), 58.6 (~~~~~~), 48.8 (pm-), 29.9 (--m--), 14.8 (----) and 0 (- -). Ethanol concentration and acetic acid concentration in (B) are (73.4 g/l, 33.9 g/[) (-), (59.0, 32.6) (----mm), (43.7, 35.1) (pm-), (30.7, 38.8) (~~~~~),(13.6, 36.0) (-pp-) and (0, 34.5) (~ -). Ethanol concentration and acetic acid concentration in (C) are (30.8 g/l, 97.0 g/l) (-), (29.6, 75.3) (------), (30.1, 56.6) (- -), (30.7, 38.8) (~~~~~),(31.5, 20.2) (- p-) and (29.9, 0) (~ -). Acetic acid concentration (g/l) in (D) is 97.0 (-), 75.3 (mm-m-), 56.6 (pm-), 38.8 (~~~~~),20.2 (p-p-), 11.3 (-) and 0 (-).

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J. FERMENT.BIOENG., solutions, acetic acid concentration, C,,,, was almost the same. According to Fig. 3B, d210g(l/7) at 1,674, 1,706 and 1,718 nm was not affected by the addition of ethanol. The negative peaks observed at 1,672 and 1,718 nm in Fig. 3D were shifted to longer wavelengths upon adding ethanol to the acetic acid solution. Therefore, the wavelength of 1,674 nm should be selected as the first wavelength for acetic acid calibration. Calibration and validation for determining acetic acid concentration in the culture broth MLR was conducted on second-derivative spectra and C,,, for acetic acid in the culture broth. As a result, the following calibration equation for measuring acetic acid was obtained.

of 1,686 nm should be selected as the first wavelength for ethanol calibration. Calibration and validation for determining ethanol concentration in the culture broth MLR was conducted on second-derivative spectra and C,,, for ethanol in the culture broth. As a result, the following calibration for measuring ethanol was obtained. C,,,=13.04-105.2A-257.2A2 (6)

Here, Al and A2 are the values of d210g(l/Z) at 1,686 and 1,738 nm, respectively. The values of R and the standard error of calibration (SEC) were 0.999 and 0.374 g/l, respectively. Validation of the calibration equation, Eq.6, was carried out. Ethanol concentration in the prediction sample set (n=21), which was not used for calibration, was predicted using the calibration equation, Eq.6, and compared with the values of C,,,. The results are shown in Fig. 4. In the figure, the actual values, C,,,, are plotted on the horizontal axis and NIR-predicted values, C,,,, on the vertical axis. The standard error of prediction (SEP) was 0.387g/l with a bias of -0.002. Excellent agreement between the conventional method and NIR was observed with r=0.997. Correlation plots for determining acetic acid concentration in aqueous samples The values of r calculated using second-derivative spectra obtained for aqueous samples of authentic acetic acid solutions are plotted in Fig. 2B. The values of r obtained for aqueous samples of authentic acetic acid mixed with ethanol are also shown in Fig. 2B. Values of r close to -1 were observed in 9 wavelength regions in the case of authentic acetic acid solutions. Values close to - 1 were observed in 5 wavelength regions in the case of solutions of authentic acetic acid mixed with ethanol. The absolute values of r around the wavelengths of 1,178, 1,278, 1,370, 1,538 and 1,830 nm decreased considerably on adding ethanol to authentic acetic acid solution. The selection of the wavelength for the acetic acid calibration equation was examined. As shown in Fig. 30, peaks due to acetic acid were observed at 1,672 and 1,718 nm in each spectrum of the aqueous solutions of acetic acid. Spectra of aqueous mixed solutions of ethanol and acetic acid are shown in Fig. 3B. In these mixed

C,,,=

-45.1

-3515A1+3179A2

(7)

Here, A, and A2 are the values of d210g(l/T) at 1,674 and 1,718 nm, respectively. The values of R and SEC were 0.940 and 0.387 g/I, respectively. Validation of the calibration equation, Eq. 7, obtained for acetic acid was carried out. The acetic acid concentration in the prediction sample set (n=21) was predicted using Eq. 7 and compared with the values of C,,,. The results are shown in Fig. 5. The SEP was 0.248 g/l with a bias of -0.002. Good agreement between C,,, and C,,, was observed with r=0.976. Monitoring of ethanol and acetic acid concentrations in the culture broth Based on the results described above, it was concluded that NIR could provide accurate predictions of the concentrations of ethanol and acetic acid in the culture broth of a rice vinegar fermentation simultaneously. Time courses of ethanol and acetic acid concentrations in the culture broth are shown in Fig. 6. The predicted concentrations for ethanol and acetic acid are represented by closed symbols, while open symbols denote those obtained by the conventional method. Some of the culture broth was drawn out and fresh medium was added to the fermentor at 4.5 h. For both constituents, the values predicted using NIR were in good agreement with those obtained by the conventional method. The values of correlation coefficients for ethanol and acetic acid were 0.999 and 0.989, respectively. Measurement of optical density of the culture broth The absorption of visible rays between 400 and 800 nm

ov 0

10

20 C act of ethanol

30 (g/I

40
C.,,

a0
of acetic

90
acid (gl

100

110

I)

FIG. 4. Correlation between ethanol concentration, C,,, obtained using the conventional method, and that predicted using NIR with the calibration equation produced using d?log(l/T) at 1,686 and 1,738 nm, C&=13.04-105.2A,-257.2Az. Solid line represents C,,,=C,,,. The values of SEP, bias and r were 0.387 g/f, -0.002 and 0.997, respectively.

FIG. 5. Correlation between acetic acid concentration, C,,,, obtained using the conventional method, and that predicted using NIR with the calibration equation produced using dlog(l/ZJ at 1,672 and l,718nm, CD,,=-45.1-3515A,+3179AZ. Solid line represents C,,,=C,,. The values of SEP, bias and r were 0.248 g/l, -0.002 and 0.976, respectively.

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after the analysis, because NIR is a nondestructive method. The results of the present study suggest that NIR may be a useful method for the monitoring and control of rice vinegar fermentation. If the concentrations of other constituents, such as organic acids, in the culture broth can be measured simultaneously, NIR will become over more important in the management of the vinegar fermentation process. Prediction of the concentrations of other constituents in the culture broth of rice vinegar fermentation should be studied in the future.
ACKNOWLEDGMENT

_-I

4
Time (h)

FIG. 6. Time courses of ethanol and acetic acid concentrations in the rice vinegar fermentation. Some of the culture broth was removed and fresh medium was added to the fermentor at 4.5 h. Symbols: 0, C,,, of ethanol; 0, C,, of ethanol; a, C,,, of acetic acid; A, C,,, of acetic acid.

The authors are very grateful for the Hiroshima City University Grant for Special Academic Research (1995 and 1996) and to The Iijima Memorial Foundation for the Promotion of Food Science and Technology (1995) for funding this study.
REFERENCES 1. Stark,

by the same culture broth as used for determining the calibration equation of ethanol was measured using the near-infrared spectrophotometer. The optical density of the samples was distributed in the range from 0.15 to 0.33. On comparing the values of the optical density of the culture broth measured using the near-infrared spectrophotometer with those measured at 660nm using the spectrophotometer in the conventional analysis, a very good correlation with r over 0.99 was observed at each wavelength between 488 and 800nm (data not shown here). Therefore, it is possible to measure the optical density of the culture broth at the same time as the measurement of ethanol and acetic acid concentrations. Gas chromatography is applied to Total discussion the measurement of the concentrations of ethanol and acetic acid in the culture broth of a vinegar fermentation. The operational procedure of gas chromatography is simple and the time required for the measurement is about 5 min per sample. Automatic on-line measurement can be achieved using a gas chromatograph equipped with an automatic injector. For NIR, while, the optical density and the concentrations of ethanol and acetic acid in the culture broth could be simultaneously analyzed. The operational procedure of NIR was also very simple and the time required for the measurement was only 5 min. In addition, it is possible to transport the culture broth back from the spectrophotometer to the fermentor

2.

3. 4.

5.

E., Luchter, K., and Margoshes, M.: Near-infrared analysis (NIRA): a technology for quantitative and qualitative analysis. Appl. Spectrosc. Rev., 22, 335-399 (1986). Dumoulin, E. D., Azais, B. P., and Guerain, J. T.: Determination of sugar and ethanol content in aqueous products of molasses distilleries by near infrared spectrophotometry. J. Food Science, 52, 626-630 (1987). Kaffka, K. J. and Norris, K. H.: Rapid instrumental analysis of composition of wine. Acta Alimenta., 5, 267-279 (1976). Coventry, A. G. and Hunston, M. J.: Application of nearinfrared spectroscopy to analysis of beer samples. Cereal Foods World, 29, 715-718 (1984). Halsey, S. A.: The use of transmission and transflectance near infrared spectroscopy for the analysis of beer. J. Inst. Brew., 91, 306-312 (1985).

M.: Prediction of the concentration of 6. Yano, T. and Harata, several constituents in a mouse-mouse hybridoma culture by near infrared spectroscopy. J. Ferment. Bioeng., 77, 659-662

(1994).
7. Mark, H.: An analysis of near-infrared data transformations, p. 55-68. In Patonay, G. (ed.), Advances in near-infrared measurements. JAI Press, London (1993). M.: Nondestructive quality measurement of food by 8. Iwamoto, a near infrared spectroscopic technique. Nippon Shokuhin KOEYO Gakkaishi. 27(9), 464-472 (1980). (in Jauanese) B. G. and k&n, T.: Near inf;aredzspectrbscopy in 9. O&me, food analysis, p.86-103. John Wiley & Sons, New York (1986). 10. Osborne, B. G., Fearn, T., and Hindle, P. H.: Practical NIR spectroscopy with applications in food and beverage analysis, p. 13-35. Longman Scientific & Technical, Harlow (1986).