a rapid spectrophotometric method for quantitative

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A rapid spectrophotometric method for quantitativedetermination of lactulose in heated milk and milk

productsAk Adhikari, D Sahai, On Mathur

To cite this version:Ak Adhikari, D Sahai, On Mathur. A rapid spectrophotometric method for quantitative determinationof lactulose in heated milk and milk products. Le Lait, INRA Editions, 1991, 71 (5), pp.555-564. �hal-00929267�

https://hal.archives-ouvertes.fr/hal-00929267

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Lait (1991) 71,555-564© Elsevier/INRA

555Original article

A rapid spectrophotometric methodfor quantitative determination of lactulose

in heated rnllk and rnllk products

AK Adhikari, 0 Sahai, ON Mathur

National Dairy Research Institute, Division of Dairy Chemistry, Kamal (Haryana), India 132001

(Received 20 August 1990; accepted 20 February 1991)

Summary - The reddish-brown colour produced on heating protein-free filtrate of heated milkscontaining less than 1.0% lactose and free of ions in 6.4 N sulphuric acid was used for quantitativedetermination of lactulose. The absorptivity of the coloured solution of lactulose at 330 nm was 14-fold more than that obtained from 62.S-fold more concentration of lactose and other aldoses. By fol-lowing the optimum conditions established in this method, a minimum of 10 mg/100 ml concentrationof laetulose can be determined in high heated unsweetened milk and milk products. The methodwas found to be very useful and sufficiently sensitive under dairy industry conditions where the ionremoved step can be omitted if raw milk filtrate is taken for blank preparation.

lactulose 1carbohydrate 1 heat treatment 1dried milk 1 UHT milk

Résumé - Méthode rapide de dosage par spectrophotométrie du lactulose dans le lait et lesproduits laitiers chauffés. La méthode de dosage du lactulose proposée est basée sur sa proprié-té à produire une coloration rouge brune au cours du chauffage (100 oC - 20 min) en milieu sulfu-rique (6,4 N). L'absorption de la solution de lactulose colorée à 330 nm est 14 fois supérieure à celled'une solution de lactose et d'autres aldoses dont la concentration est 62,5 fois supérieure. Cetteméthode permet, en suivant les conditions optimales établies, de déterminer une concentration mini-male de 10 mgl100 ml de lactulose dans des laits et produits laitiers non sucrés ayant subi un forttraitement thermique. Elle est en outre très utile et suffisamment sensible à l'échelle industrielle oùl'étape de désionisation peut être supprimée si le filtrat de lait cru est pris comme blanc.

lactulose 1 hydrate de carbone 1 traitement thermique 1poudre de lait liait UHT

556

methylamine method of Nikerson et al(1976) is not very useful in milk where thelactose content is much higher than that oflactulose. The spectrophotometric methodof Parrish et al (1980) is time-consumingand requires separate determination of ailcarbohydrates.

This paper describes a simple and rapidspectrophotometric method which does notrequire separation of lactulose from lac-tose or other aldoses and can be conven-iently applied for quantification of lactulosein unsweetened milk and milk products.

AK Adhikari et al

INTRODUCTION

Lactulose (4-0- B-D-galactopyranosyl-D-fructo-furanose) is formed in heated milksby the alkaline isomerization of lactosecatalysed by the free amino group of case-in (Richards and Chandrasekhara, 1960;Adachi and Patton, 1961). Lactulose oc-curs in 2 forms: as lactulose in free solu-tion, and as N-deoxylactulosyl-L-Iysinewhich is covalently bound to the aminogroup of the milk proteins (Henry et al,1948).

The formation of lactulose in heatedmilks is dependent on time and tempera-ture of heating and is also significantlyaffected by the pH of the environment(Adachi, 1959; Martinez-Castro and Ola-no, 1980). The lactose concentration inraw milk plays an important role in the for-mation of lactulose (Andrews, 1989).

Several methods have been used forthe determination and quantification of lac-tulose in milk and milk products. The majorproblem with quantification of lactulosearises from the fact that the concentrationof lactose is far in excess of that in milkand milk products. Most of the methodstherefore include preliminary separation ofcarbohydrate mixtures or application of aspecific aldose reaction to rem ove the lac-tose and thereafter quantification of lactu-lose either by a general carbohydratemethod or by TLC, GLC or HPLC meth-ods. Such methods are not only cumber-some and expensive, but also time-consuming when a large number of sam-pies have to be analysed.

The spectrophotornetrlc method ofAdachi (1965) utilizes a lengthy procedureof oxidation of aldoses to aldonic acid byhypoiodate, whereas the anthrone methodof Zagrodzki et al (1968) is reported togive slightly higher values as anthronereacts with 5-hydroxymethylfurfural. The

MATE RIALS AND METHODS

The principle of this method is based on the factthat ketoses are decomposed far more easilythan aldoses by the action of minerai acidswhich produces an intense red or violet coloura-tion, while the colour appears relatively slowlyand is less intense when minerai acids act uponaldoses (Stanèk et al, 1963). As heated milkscontain no ketose other than lactulose (Gordonet al, 1956), the principle applied in this methodis specifie and consistent.

Mater/ais

AR grade lactulose (98.0%) was obtained fromSigma: Chemical Co, USA. 6.4 N H2S04 (ARgrade) and 40.0% TCA (AR grade) solutionwere freshly prepared before use. Amberlite IR-120 and Amberlite IR-458 (Sigma Chemical)were used as cation and anion exchanger re-spectively. HCI 3 N and NaOH 2 N (AR grade)were used for washing the column.

Standard methods usedfor comparative study

Lactulose was separated by TLC method ofMartinez-Castro and Dlano (1981) followed byquantification by the method of Adachi (1965)and cysteine-hydrochloride-carbazole colorimet-

Spectrophotometric determination of lactulose 557

ric method of Fujita and Iwatake (1931). The re-sults obtained were compared with the H2S04method. Total carbohydrate content in the milkfiltrate was determined by the phenol-sulphuricacid method of Brich and Mwangelwa (1974).

Milk samples

UHT milk samples were obtained from a com-mercial milk plant (indirect type) which had beenprocessed at 140 °C/3 s. Sterilized (120 °C/15min) and boiled milk (10 min) samples were pre-pared in the laboratory from fresh cow's milk.Skim milk powders (roller- and spray-dried) werecollected from the experimental dairy at the Na-tional Oairy Research Institute, Karnal, India.

Method

Ten ml of warmed milk (37 oC) was treated with40% TCA solution and kept for 10 min to precipi-tate out the total proteins, and the final volumewas made up to 100 ml (dilution coefficient 10-1)with the same TCA solution. The whole contentwas filtered using Whatman No 42 filter paper.

Removal of interfering ions

To remove the interfering ions (cations Ca2+,Mg2+, Na+, K+ and anions Cit3-, HPO~+, HC0:ï,SO~- and RCOO- and their complexes; Holt etal, 1981), the filtrate was passed through a cat-ion exchange column then an anion exchangecolumn in free base according to the procedureof Adachi (1965). The cation exchange columnof Amberlite IR-120 in sodium form and the an-ion exchange column of Amberlite IR-458 werefilled to 10 cm height in a 45-cm high 18-mmdiameter column with glass wool and a flow con-trol device. The former was treated with 100 mlof 3 N HCI and then washed with distilled wateruntil the effluent showed a negative chloridetest. The latter was treated with 100 ml of 2 NNaOH and th en rinsed with water until the efflu-ent showed a negative base test with phenol-phthalein indicator. The total milk filtrate wasthen allowed to pass successively through the 2columns at the rate of 4-5 ml per min, followedby washing with 100-110 ml of distilled water.

The volume of the effluent was adjusted to 100ml by removing excess water under vacuum at65-70 -c.

Development of colour

Five ml of the above effluent was taken into aPyrex 15 x 1.5 cm-test tube fitted with a glassstopper treated with 5.0 ml of 6.4 N H2S04 andplaced into a constantly boiling water bath(100 ± 0.5 "C) for 20 min. The tube was cooledimmediately to 37 ± 2 oC and absorbance wasmeasured within 30 min at 330 nm by a spectro-photometer (Spectronic 200) against a blankprepared in a similar manner but with 5 ml of40% TCA solution in place of the heated milk fil-trate. For dried unsweetened milk powders,1.0 9 of the sam pie was dissolved in 10 ml ofwarm distilled water, diluted to 100 ml with 40%TCA solution (dilution coefficient 10-2) and theabove procedure was followed for quantification.

Calibration curve

The calibration curve for lactulose was preparedby taking standard lactulose solution (conc 0.2-1.2 mg/5 ml) and developing the colour in a si mi-lar manner as for the milk filtrate.

RESUL TS AND DISCUSSION

The action of minerai acids on sugars ledto a number of decomposition products,sorne of which were coloured (Stanèk etal, 1963). Besides a certain amount of hu-mus-type decomposition, both aldohex-oses and ketohexoses yield HMF as themain intermediate, which on further de-composition with acid produces furfural, acolouring substance.

Absorbance spectm (À maxJ

Absorbance spectra of standard lactulosesolution (conc 2.92 x 10-3 molli) was ex-

558 AK Adhikari et al

amined in the range of 310-345 nm wave-, length by a Spectronic 20 0 spectropho-

tometer (fig 1). The absorbance maxima(Âmax) was slightly broad and noted at 330nm of 0.616 x 102 molli absorptivity.

EffectofconcenuaUonofacidon cotour deve/opment

The effect of the strength of sulphuric acidon lactulose and other aldoses after heat-ing 20 min in a constantly boiling waterbath (100 ± 0.5 OC) on colour developmentis depicted in figure 2. It was observed thatup to an acid concentration of 6.4 N, col-our development for 1.0% lactose, glucoseand galactose solution was significantlyless (00 $ 0.01), whereas 0.016% solution

0.16

0.16

0.14

~ 0.12

1~ 0.10

8·0.06

0.06

0.04

0.02

315 320 325 330

Wavelength (nm)

Fig 1. Absorption spectra of lactulose (2.92 x10-3 molli) in 6.4 N H2S04 at different wave-lengths.Spectre d'absorption du lactulose (2,92 x 1o-JmollI) dans HzS04 6,4 N, à différentes lon-gueurs d'ondes.

of lactulose gave nearly 14 times more col-ouration (00 == 0.14). This reveals that thelactulose concentration of 62.5 times lessthan lactose and other aldoses produces14 times more colouration with sufficientspecificity.

Effect of the /ength of heaUng Umeon cotour deve/opment

Rapid colour development was observedin the initial 20 min of heating of lactulosesolution in 6.4 N H2S04, whereas the col-our development for other aldoses wasless (s 0.012 OD) than that for lactulose fora similar extent of heat treatment (fig 3).Lactulose concentration of 0.02% produc-es 14 times more colouration (OD == 0.178)

0.16

0.16

EgO.14

'":;;~0.12

]~0.10

80.06

0.06

O.Qo\

5.6 5.6 6.0 6.2 6.4 6.6 6.6Concentration of H1SO. (N)

Fig 2. Effect of strength of H2S04 on the colourdevelopment of lactulose and other aldoses. a:lactulose (0.016%), b: lactose (1.0%), c: glu-cose (1.0%), d: galactose (1.0%).Effet de la concentration en H~04 sur l'intensi-té de la coloration produite par le lactulose etd'autres aldoses. a: lactulose (0,016%), b: lac-tose (1,0%), c: glucose (1,0%), d: galactose(1,0%).


0.18

0.16

0.14

~li! 0.12

";;~ 0.10

1rOB0.06

0.04

0.02

10 15 20 25 30 35lime of heating (min)

Fig 3. Effect of length of heating time on the co-lour development of lactulose and other aldoses.a: lactulose (0.02%), b: lactose (1.0%), c: glu-cose (1.0%), d: galactose (1.0%).Effet de la durée du temps de chauffage sur J'in-tensité de la coloration produite par le lactuloseet d'autres aldoses. a: lactulose (0,02%), b: lac-tose (1,0%), c: glucose (1,0%), d: galactose(1,0%).

than lactose and other aldoses at 1% con-centration (00 s 0.012).

Calibration curve for /actu/ose

The plot of absorbance versus lactuloseconcentration (0.2-1.2 mg/5 ml) was linearat 330 nm and obeyed the Beer-Lambert'slaw in this range. The concentration of theacid used and the time of heating were im-portant determinant factors in the linearityof the curve. The higher the acid concen-tration and the time of heating, the greaterthe irregularities in the colour developmentobserved. The regression equation devel-oped for lactulose concentration (Y = mglactulose/5 ml) and absorbance (X = ob-served optical density) was calculated as:

y = 28.50 X - 0.10

cotour contribution of different a/dosesin relation to /actu/ose equiva/ent

Table 1 shows the relative colour develop-ment of lactose, glucose and galactose at

Table 1.Colour contribution of different aldoses with respect to lactulose equivalent.Absorption à 330 nm du lactose du glucose et du galactose en fonction de leur concentration dans lefiltrat.

Concentration (%)in the filtrate

Absorbance at 330 nm

Interfering aldosesLactose Glucose Galactose

0.000 ( 0.00)* 0.000 (0.00) 0.000 ( 0.00)0.001 ( 0.00) 0.000 (0.00) 0.001 ( 0.00)0.004 ( 0.18) 0.002 (0.00) 0.003 ( 0.00)0.006 ( 1.32) 0.004 (0.18) 1 0.007 ( 1.89)0.018 ( 8.16) 0.009 (3.03) 0.016 ( 7.02)0.039 (20.12) 0.021 (9.89) 0.029 (14.43)

0.500.751.001.251.502.00

* Figures in parentheses are mg equivalent lactulose per 100 ml 01 Iiltrate.Les chiffres entre parenthèses représentent des mg d'équivalent lactulose pour 100 ml de filtrat.

560

Relative weight equivalentof aldoses (mg/tOO ml)

AK Adhikari et al

Table Il. Effect of intertering aldoses on colour contribution at the same absorbance value.Poids relatif des différents aldoses pour une même valeur d'absorption.

Lactulose(mg/tOO ml filtrate)

Absorbanceat330nm

Lactose Glucose Galactose

1020

0.020.04 2000

various concentration in relation to mgequivalent lactulose/100 ml of milk filtrate.lt is evident from the table that 1.0% of lac-tose, galactose and glucose in milk filtratecontribute to the similar coiour intensity of'" 0.18, 0.00 and 0.00 mg of lactulose per100 ml in the respective milk filtrates. Thisc1earlyreveals that the intensity of colourcontribution by aldoses is much less thanthat of the colour developed by lactuloseeven up to the 1.0% level. Table Il repre-sents the colour contribution of mg equiva-lent weight of lactulose in comparison tothe mg equivalent of aldoses. lt is evidentfrom the results that '" 100, 180 and 200times weight equivalent of lactose, galac-tose and glucose contribute a similar inten-sity of colouration to that of lactulose onfollowing the optimum conditions pre-scribed in this method.

Recovery studies

Recovery studies were carried out by add-ing standard lactulose solution in heatedmilk and milk filtrate (in case of milk prod-ucts). The results obtained are presentedin table III. Appraximately 97-101% recov-ery was observed, indicating high accura-cy and reproducibility of the method devel-oped.

2000 1800

Effect of anion exchange resinon reducing sugars and raw mi/k fi/trate

The effect of anion exchange resin on re-ducing sugars is presented in table V. Theresult shows that on passing 1% lactosesolution through the anion exchange resinthere was a negligible increase in opticaldensity at 330 nm. The initial value of0.010 increased to 0.013 on passing 1%lactose solution, while the initial value of0.181 dropped slightly to 0.174 after pass-ing the lactulose solution (conc 1 mg/ml)through the anion exchange resin. At thesame time when the raw milk filtrate was

Table III. Percentage of recovery of lactuloseadded to milk/milk filtrate.Pourcentage de récupération du lactulose ajou-té au lait/filtrat de lait.

Lactuloseadded

(mg/Sml)

% RecoveredLactuloserecovered(mg/Sml)

1.02.03.04.0

97.79101.61100.0497.83

0.982.033.003.91


Table IV. Lactulose content of heated milk and milk products - comparison of 3 different methods.Teneur en lactulose de laits et produits laitiers chauffés. Comparaison de 3méthodes différentes.

Sample Lactulose (mg/100 g)

nc method Cysteine - HCI- carbazole method H:zS04 method

Boiled milk (10 min) 84.24 (2.50)" 83.02 (2.75) 85.40 (2.55)Sterilized milk (15 min) 166.77 (2.22) 162.60 (1.80) 165.24 (2.08)UHT milk 58.60 (1.80) 57.24 (1.35) 56.04 (1.56)(indirect type)SMP (roller-dried) 478.28 (2.90) 474.30 (2.75) 479.80 (2.90)SMP (spray-dried) 266.17 (2.05) 261.40 (2.25) 268.34 (2.60)

" Figures in parentheses are standard deviations.

Table V. Effect of anion exchange resin on reducing sugars (le lactose and lactulose) and raw milk fil-trate.Effet de la résine échangeuse d'anions sur l'absorption des sucres réducteurs (lactose et lactulose) etdu filtrat de lait cru.

passed through the anion exchanger. theinitial value of 0.153 dropped sharply to0.028 due to removal of intertering ionsand the subsequent colour developmentwas very insignificant in contributing to lac-tulose value. This reveals that neither werethe reducing sugars adsorbed nor was thelactose isomerized to lactulose while pass-ing through the anion exchanger. Turtonand Pacsu (1955) observed a negligible

change in mutarotation and in the nature ofthe sugar solution (2%) when passedthrough an anion exchange resin IRA-400-OH. However, they observed a rapid ad-sorption of o-çlucose, D-fructose and D-mannose in the hydroxide form of resin; D-fructose was transformed to o-psicose, butno evidence was found of changes in re-ducing sugars Iike lactose or lactulose.Adachi (1965) reported no adsorption or

Raw milk filtrate Standard lactulose solution Added lactulose (mg/5 ml)(cane 1mg/ml)

Untreated Treated Untreated Treated Untreated Treated Practical Determined % loss

OD0.010 0.010 0.155 0.030 0.185 0.178 5.0 4.86 2.800.005 0.D10 0.160 0.035 0.178 0.1700.015 0.020 0.145. 0.020 0.180 0.175

Mean 0.010 0.013 0.153 0.028 0.181 0.174

Standard lactosesolution (1%)


isomerization of lactulose while passing itthrough strong cation and anion exchangeresins for a long time to remove interferingions and thereafter successfully deter-mined lactulose content spectrophotomet-rically. Our findings also showed a negligi-ble adsorption of lactulose and noisomerisation of lactose to lactulose whilepassing through the anion exchange resin.This might be due to the strong acidic en-vironment present in resin provided by themilk filtrate containing 40% TCA solution.

Omission of the ion exchange step:effect of changes in absorbancewith raw mi/k fi/trate and additionof /actu/ose solution

To study the efficacy of the method byomitting the ion exchange step, raw milkfiltrate replaced heated milk filtrate and thelactulose solution was added at an in-creasing concentration, (fig 4) with the

0.18

ê0

'"'"1;.~ 0.10

ii0.08

0.08

0.04

0.02

0.18

0.2 0.4 0.6 0.8 1.0 1.2

Fig 4. Calibration curve for lactulose (conc 1.16x 10-4 to 7.01 X 10-4 mol/l).Courbe de calibration pour le lactulose (conc1,16x 10-4-7,01 x 10-4molli).

same quantity of raw milk filtrate to makeup the total volume to 5 ml. Both the sys-tems were treated in a similar manner forcolour development as depicted in figures5 and 6. The Iinearity of both the slopescurves reveals that they obeyed the Beer-Lambert's law accurately in the given con-centration range of lactulose and raw milkfiltrate. This signifies the sensitivity and ac-curacy of the method, provided the rawmilk filtrate is used in place of 40% TCAsolution needed for the preparation of theblank.

Efficacy ln the determlnatlon of tectu-Jose content by uslng raw m//k fi/trateasb/ank

Table VI shows the values of lactuloseconcentration added and determined inraw milk filtrate when the same raw milk fil-trate concentrations were used for blankpreparation. The results reveal an accura-

0.24

0.22

0.20

0.18

0.18

ê~ 0.14

1ii 012f 0"10

~ 0.088

0.08

0.04

0.02

o.oo"----~~~~~~~~~~~~o

Raw milk filtrate (ml)

Fig 5. Absorbance of raw milk filtrate.Absorption de filtrat de lait cru.

0.45

0.55

0.40Eg 0.35

'"di 0.30.~

~ 0.25

} 020

0.15

0.10

0.05


8 12 18 20Lactuloee added (mg/5 ml)

Fig 6. Absorbance of laclulose solution healedwith raw milk filtrate.Absorption de la solution de lactulose chaufféeavec un filtrat de lait cru.

cy of 101.85% (mean value) in determiningthe lactulose concentration when raw milkfiltrate was taken for blank preparation in-stead of 40% TCA solution. Slightly higher

values may be attributed towards the effectof interfering ions, lactose and aldoses,which might have sorne influence on col-our development in this context.

Comparlson with standard methods

The method developed was compared withthe procedure described by Martinez-Castro and Olano (1981) and the cysteine-hydrochloride-carbazole method of Fujitaand Iwatake (1931). The results obtained(table IV) by the method developed werecomparable, consistent and reproduciblewith sufficient accuracy (standard deviationof 2.9) as compared to the other 2 meth-ods. The slightly higher values obtained formilk powders may be attributed to the high-er pre-heating temperatures followed un-der Indian conditions.

CONCLUSIONS

The rapid and simple colorimetrie methoddeveloped can successfully be performedwithin 2.5 to 3 h for routine quantitative de-termination of lactulose in unsweetened

Table VI. Lactulose content in raw milk flltrate, when raw milk filtrale was taken for blank preparation.Détermination des teneurs en lactulose ajouté dans un filtrat de lait cru (filtrat de lait cru pris commeblanc).

Vol raw milk Vollactulose Lactulose content (mg/5 ml) % Accuracyfiltrate taken (ml) solution added (ml) (b/a x 100)

Added(a) Determined (b)

1 4 16 16.28 101.752 3 12 12.44 103.663 2 8 8.28 103.504 1 4 3.94 98.505 0 0 0.24

Mean 101.85


heated milk and milk products, and is alsohighly suitable under dairy industry condi-tions where raw milk is readily available.The ion exchange step can be omitted pro-vided that the raw milk filtrate is taken forblank preparation and the colour devel-oped in a similar manner to the sample.Hence a large number of samples canconveniently be analysed for their lactu-lose content to determine the severity ofheat treatment with sufficient accuracy un-der dairy industry conditions.

REFERENCES

Adachi S (1959) The mechanism of the degra-dation of lactose in strongly heated milk.15th Int Dairy Congr, London, 3, 1686-1691

Adachi S (1965) Spectrophotometric determina-tion of lactulose with methylamine. AnalChem 37,896-898

Adachi S, Patton S (1961) Presence and signifi-cance of lactulose in milk products: a review.J Dairy Sei 44, 1375-1393

Andrews G (1989) Lactulose in heated milk. Bulltnt Dairy Fed 238, 45-52

Brich GG, Mwangelwa OM (1974) Colorimetricdetermination of sugars in sweetened con-densed milk products. J Sei Food Agric 25,1355-1362

Fujita A, Iwatake D (1931) Biochem J 242, 43(cited at the 15th Int Dairy Congr; Adachi S(1959) 3, 1686-1691)

Gordon HT, Thornburg W, Werum LN (1956)Rapid paper chromatography of carbohy-

drates and related compounds. Anal Chem28,849-863

Henry KM, Kon SK, Lea CH, White JC (1948)Deterioration on storage of dried skim milk.J Dairy Res 15, 292-305

Holt C, Dalgleish DG, Jenness R (1981) Calcu-lation of the ion equilibrium in milk diffusateand comparison with experiment. Anal Bio-chem113,154-163

Martinez-Castro l, Olano A (1980) Influence ofthermal processing on carbohydrate compo-sition of milk: formation of epilactose. Mi/ch-wissenschaft35, 5-8

Martinez-Castro l, Olano A (1981) Ready detec-tion of small amounts of lactulose in dairyproducts by thin-layer chromatography. Chro-matographia 14, 621-622

Nickerson TA, Vujicic IF, Lin AY (1976) Colori-metric estimation of lactose and its hydrolyticproducts. J Dairy Sei 59, 386-390

Parrish FW, Hicks K, Doner L (1980) Analysis oflactulose preparations by spectrophotometricand high performance liquid chromatographicmethod. J Dairy Sei 63,1809-1814

Richards EL, Chandrasekhara MR (1960)Chemical changes in dried skim milk duringstorage. J Dairy Res 27, 59-66

Stanèk J, Cerny M, Kocourek J, Pacâk J (1963)The Monosaccharide. Academic Press, NY,871-872

Turton CN, Pacsu E (1955) The effect of anionexchange resins on reducing sugars. J AmChem SocTi, 1059-1061

Zagrodzki S, Kulagowska A, Krol BW (1968)Qualitas Plantarum Mat Veg 18, 102-112 (cit-ed in Andrews GR (1986) J Dairy Res For-mation and occurrence of lactulose in heatedmilks. 53, 665-680)

a rapid spectrophotometric method for quantitative

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