264 ANALYTICAL PROCEEDINGS, JULY 1989, VOL 26

Fermentation of Cheese Whey-Monitoring by FT - IR

P. Fairbrother, W. 0. George and J. M. Williams Department of Science and Chemical Engineering, Polytechnic of Wales, Pontypridd, Mid Glamorgan CF37 1DL

The production of cheese in the world has been steadily rising. Cheese is made by adding rennin to milk to coagulate the curds which are separated off to ripen as cheese. The liquid remaining is cheese whey, which amounts to about 83% of the volume of the milk feedstock. The composition of whey depends on the type of cheese being manufactured but is typically’ about 5% lactose with fat, protein and other materials amounting to 2-3%, with water as the remainder. In previous times whey has either been fed to pigs or disposed of into the environment: it has a high BOD (35000- 40 000 p.p.m.2). With large factory-type cheese plants concern for environmental impact and appreciation of the unique nutritional and functional properties has prompted research into whey modification and utilisation. In 1955, the dairy industry processed 25% of the liquid whey produced, but by 1975, 75% was processed to utilise its nutritional and energy content. The uses included the production of liquid fuel and biogas and the growing and harvesting of yeast,3 and in the production of speciality products including lactic and citric acids, lysine, threonine, fats, xanthan gum, vitamins, enzymes and antibiotics.4 There have also been substantial uses of whey in the food industry.“.“

Alcohol Fermentation Ethanol is formed by yeast cells as a means of regenerating oxidised pyridine nucleotides in the absence of oxygen. The term “industrial fermentation” is applied to any large-scale cultivation of microbes, including those processes which are aerobic.’ Currently, 75% of bioethanol is produced by batch, stirred tank methods. In general, the investment cost of batch

methods and control devices is low but the manpower costs are high. The short time of fermentation decreases the risk of contamination but reduces the economic viability. In a semi-continuous process the medium is only partly removed and the residue serves as inoculum for the next run. In both batch and semi-continuous processes the yield of ethanol is low. In a fully continuous process there is continuous addition of nutrient and continuous removal of cells and fermenter medium. The initial change in concentrations is similar to the batch process but a steady state is soon achieved.

Yeast Strain Maiorella and Castillos evaluated an industrial batch process for producing ethanol from cheese whey using Candida pseudotropicalis ATCC 8619. The scale of operation was 250001 of whey per day, corresponding to the output of a typical cheese plant. It was estimated that the plant would yield 720 1 d-1 of 190 degree proof ethanol and 40 kg d-1 dry mass of Candida pseudotropicalis yeast. This would reduce the BOD of effluent by 90% from 35 000 p.p.m. to 3500p.p.m. at a net cost of $9.5 per 1000 1 of whey and would add 7.3 US cents per kg to the cost of cheese. This strain was used in the present work although a number of other strains have been found to be effective in particular circumstances.”10

Materials Cheese whey was obtained from Castle Dairies Ltd., Caer- philly; lactose, ethanol, sulphuric acid, sodium hydroxide, ammonium sulphate and ammonium chloride from BDH;

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ANALYTICAL PROCEEDINGS, JULY 1989, VOL 26 265

Fig. 1. solution

Photograph of arrangement of FT spectrometer with cell linked to on-line system for circulating fermentation liquid or calibration

yeast extract from Difco Laboratories, Detroit, Michigan; and Candida pseudotropicalis ATCC 8619 from American Type Culture Collection, Maryland, USA. The pre-treatment of cheese whey and the preparation of the starter cultures will be described in more detail elsewhere.11

Spectroscopic Methods All samples were measured in a Spectra-Tech multiple internal reflection “Circle Cell,” arranged for continuous flow measurements within the cell compartment of a Digilab FTS 50 Fourier Transform IR Spectrometer, using the QUANT 32 software for quantitative analysis. For recording a water background spectrum and calibration solutions a 500 ml detachable flask was connected to the cell via a circulatory peristaltic pump. In the spectrum of lactose two strong bands occur at 1076 and 1042cm-1, with weaker bands at 1156,1119 and 998cm-1. In ethanol a strong band occurs at 1046cm-1 with a weaker band at 1085 cm-1. The QUANT 32 programme was used to measure the areas between specific intervals of all seven bands. For recording samples during fermentation the flask was replaced by the fermenter vessel. The configuration of the fermenter, calibration vessel, cell and F T spectrometer are shown in Fig. 1. Spectra were scanned in the range 1300-950 cm-1. Sixty-four repeat scans were averaged for calibration samples. The resolution was 8 cm-1 at a noise level of 0.01 using triangular apodisation.

In parallel with changes in ethanol and lactose concentra- tion , growth curves for Candida pseudotropicalis were deter-

mined from measurements of absorbance at 550nm, using a Bausch and Lomb, Spectronic 501, ultraviolet - visible spec- trometer.

“On-Line” Fermentation Analysis A 900-cm3 sample of sterile, partially deproteinised whey (PDW) (pH 4.57) was transferred to the fermenter, which is shown in Fig. 1, and the heater stirrer set to 30 degrees. The whey was inoculated, aseptically, with 10 cm3 of starter culture using a sterile syringe via an inlet port at the top of the fermenter. The stirrer and peristaltic pump were switched on leading to a continuous flow of medium from the fermenter through the flow cell and back to the fermenter.

Following inoculation a computer program was initiated which recorded, saved, and plotted spectra every 30min for 24 h (Fig. 2). At the end of the period the stored spectra were analysed.

Calibration of Spectra of Cheese Whey by the K Matrix Approach If the absorptivity, a, and the path length, b, are combined into a single constant, K , then

The Beer Law expression then takes the form

If the absorbance, A l , for a mixture of components of concentrations, c1 , c2, . . . c,, is assumed from Beer’s Law to be

K = a b . . . . . . * f (1)

A = K c . . . . . . . . (2)

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266

~~~~ ~

Fig. 2. Repeat scans of fermentation mixture at 30-min intervals over 24 h between 950 and 1300 cm-1. Note the diminution of lactose bands at 998, 1042, 1076, 1119 and 1156 cm-l and the growth of ethanol bands at 1046 and 1085 cm-I

additive for a particular wavenumber value, 1, A1 = klcl + k2~2 + ... + k,c,, . . . . (3)

In general, (2) is a matrix equation and in the case of a two-component mixture such as ethanol and lactose the matrix can take a simple form

(4)

where c1 is the concentration of ethanol and c2 is the concentration of lactose. Absorbance measurements are made at two wavenumber values, A l and A2. If the two bands are not overlapped then:

In general, absorption bands overlap and the entries in the K matrix are non-zero. These entries can be computed by over-determination of calibration data by measurement of a larger number of standards and wavenumber values than the minimum needed (two in the present work). The concentration values of the standards and the wavenumber values at which measurements are made are chosen to optimise the results of a particular unknown systern.I2

The K matrix is determined within the QUANT 32 package by a least-squares regression based on seven measured absorbances on each of 13 solutions of known ethanol and lactose composition as the calibration step. The matrix is then used to calculate concentrations of fermentation liquid from measured absorbances as the analytical step.

Precision and Accuracy A printout of the calculated values for the 13 standard mixtures is given in Table 1, which shows a root mean square error of 0.1059 and 0.1196 when compared with known values for ethanol and lactose, respectively. This gives an indication of precision of the on-line fermentation results. The accuracy would be dependent on the nature of other components present in the sample but absent from the standards. In principle, subtraction of standard bands from sample bands would show bands from additional components. It follows that using QuAN.r 32, accuracy is effectively limited by the quantitative physical performance of the instrument and some knowledge of the components present in sample and standards.

ANALYTICAL PROCEEDINGS, JULY 1989, VOL 26

Results Fermentations were carried out by using 10 and 25cm3 of inocula with and without 1% supplements of yeast extract and ammonium sulphate or chloride. Typical representations of results from a particular run are shown in graphical form in Fig. 3, but may also be printed out in tabular form. In terms of previous considerations it would be possible to pursue greater accuracy, but in terms of needs for the purpose of the present on-line monitoring work the results have reasonable reliability and enable the following conclusions to be drawn.

~~

Table 1. The composition of calibration mixtures and the back calculation of concentration of each mixture as an indication of precision

Ethanol concentration, %mlV Lactose concentration, %mlV

Actual Measured Error Actual Measured Error 4.1240 4.1590 -0.0350 0.1500 0.1834 -0.0334 3.9650 4.1518 -0.1868 0.2000 0.1362 0.0638 3.3310 3.2026 0.1284 0.7500 0.7406 0.0094 3.1720 3.0111 0.1609 1.0000 1.0204 -0.0204 2.8550 2.9712 -0.1162 1.2000 1.1600 0.0400 2.5380 2.4265 0.1115 2.0000 1.8791 0.1209 2.2200 2.1951 0.0249 2.5000 2.6848 -0.3848 2.0620 2.0286 0.0334 3.0000 3.1210 -0.1210 1.5070 1.4976 0.0094 3.7500 3.7160 0.0340 1.1100 1.1823 -0.0723 4.0000 3.7857 0.2143 0.9520 0.9500 0.0020 4.2500 4.4751 -0.2251 0.6740 0.8287 -0.1547 4.5000 4.3690 0.1310 0.3970 0.2957 0.1013 4.7500 4.7662 -0.0162

RMS error 0.1059 RMS error 0.1 196

Conclusions Firstly, an increase in inoculum size led to an increase in the rate of ethanol production. Secondly, addition of nutrients increased the alcohol production rate, but led to a decrease in lactose utilisation and ethanol production. Thirdly, the growth of Cundidu pseudotropicalis appeared to parallel the produc- tion of alcohol. Fourthly, increased aeration led to a rise in the growth rate of the yeast, which suggests that forced aeration may lead to a decrease in fermentation time.

5

L I +-+- +.

p 4+-+-+, $ 1 +'t.

0 5 10 15 20 25 Timeih

Fig. 3. The production of ethanol and the utilisation of lactose by Cundida pseudotropiculis using a 25-cm3 inoculum: a. ethanol; + , lactose

Hence, quantitative FT - IR has proved to be an effective method for on-line analysis of the alcoholic fermentation of cheese whey. The technique could readily be transferred from a laboratory or pilot plant scale to an industrial scale with real time process control.

Future Developments-FT - IR Using an Optical Fibre? The use of a mid-infrared transmitting optical fibre for on-line analysis has recently been reported by Compton et al.13 The

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ANALYTICAL PROCEEDINGS, JULY 1989, VOL 26

~ Light Microscopy is one of the oldest techniques at the disposal of the analyst and is unfortunately greatly undervalued and underused in the analytical laboratory. It is, in fact, a conventional economical technique which should not be overlooked and can be of great value in the analysis of foods, pharmaceuticals, metals, plastics, water,

I agrochemicals, textiles and much more.

267

In this book the authors draw upon their considerable experience in industry and consulting practice, to provide examples of the many and varied uses of light microscopy in analysis. They describe in detail its capabilities and seek to encourage its wider use in actual practice, reminding analysts of its qualities and applications. They also advocate good practice in its use.

1 Microscopists, analysts and students alike will gain much from the authors’ enthusiasm and as a result may assist in extending the utility of the instrument into the future.

possibility of remote sampling by carrying the spectral informa- tion through an optical fibre opens up exciting new possi- bilities, including studies of samples which are not readily placed in the cell compartment of a spectrometer. These include hazardous materials, processes inside reactors, indus- trial furnaces and remote environmental systems.

Currently, the transmittance of optical fibres is limited to about 1200cm-1, but it is to be hoped that this will be extended further into the infrared region and make available the wealth of information in the infrared to sensors which were, hitherto, largely limited to single bulk measurements.

References 1 .

2.

3.

Kosikowski. F. V., “Cheese and Fermented Milk Products,” Edwards Brothers Inc., Ann Arbor, Michigan, 1977, p. 450. Singh. V., Hsu. C. C., and Tzeng, C. H . , Process Biochem., 1983.37, 13. Mann, E. J . , Dairy Ind. Int., 1987, 52, 12.

4. 5. 6. 7.

8.

9.

10.

11 .

12.

13.

Kosaric, N. , and Wieczorek, Dev. Food Sci., 1982, 9, 229. Mann. E. J., Dairy Ind. Int., 1988, 52, 6. Kennedy, J . P., Cultured Dairy Prod., 1985, 20, 13. Primrose, S. B., “Modern Biotechnology.” Blackwell Scien- tific Publications, Oxford, 1987, pp. 5141. Maiorella, B. L., and Castillo. F. J., Process Biochem.. 1983, 19, 157. Castillo, F. J. , Izaguirre, M. E., Michelena, V., and Moreno, B., Biotechnol. Lett., 1982, 4, 567. lzaguirre, M. E., and Castillo, F. J., Biotechnol. Lett., 1982,4, 257. Fairbrother, P., George, W. O., and Williams, J . M., In preparation. Crocombe, R. A. , Olson, M. L., and Hill, S. L., in McClure, G. L., Editor, “Computerised Quantitative Infrared Analysis, ASTM STP 934,” American Society for Testing and Materials, Philadelphia, 1987, pp. 95-130. Compton, D. A. C., Hill, S. L . , Wright. N. A. , Druy, M. A. , Piche, J . . Stevenson, A., and Vidrine, D. W . , Appl . Spec- trosc., 1988, 42. 941.

An Introduction to Applications of Light Microscopy in Analysis

By D. Simpson and W.G. Simpson, Analysis for Industry, Thorpe-Ze-soken

Price 229.50 ($63.00) ISBN 0 85186 987 4. Hardcover 215pp information

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