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SWAI N et a1.


is available for the commercial application of both of these techniquesz41 25 but the benefit to be
gained in canning of citrus juice must be regarded as doubtful.
Complete removal of oxygen from orange juice improves the retention of ascorbic acid
and flavour during processing but has little effect on the retention of these quality factors
during storage. In relation to pasteurised, canned citrus juices, the importance of the de-aera-
tion operation has been over-emphasised. Since oxygen disappears rapidly from the contents
and the atmosphere of the can during storage at ordinary temperatures, it is not profitable to
attempt to achieve highly efficient de-aeration. Removal of the bulk of the dissolved air,
particularly when the juice is supersaturated, is sufficient to confer the main benefits of better
initial flavour and ascorbic acid content, and decreased corrosion of the can.
With frozen juices, however, oxygen does not disappear rapidly from the container and
efficient de-aeration may be amply justifiable in terms of improved retention of flavour and
ascorbic acid.
The authors wish to express their sincere gratitude to Mr. G. G. Coote of the Division of
Mathematical Statistics, C.S.I.R.O., for his painstaking assistance with the statistical aspects
of this investigation, and to Mr. R. A. Edwards for analytical assistance.
Commonwealth Scientific and J ndustrial Kesearch Organisation
Division of Food Preservation and Transport
Homebush, N . S. W., Australia
1 McDermott, F. A, Bull. Fl u agvic. E,v@. Sta. , 1917,
2 Hunnikin, C., Tech. Conznzz~n. Bzir. Hovt., E.
3 Lewis, V. M., & McKenzie, H. .A,, Annalyt. Clzem.,
a Boyd, J . M., & Peterson, G. T., I ndust v. Engng
Chem. ( f i zdust r. Ed n) , 1945. 37, 370
5 Hicks, E. W., Kefford, J . F., & McKee, H. S. ,
' Report on Food Stores in New Guinea ', 1945,
p. 13 (Melbourne : Council for Scientific and
Industrial Research)
NO. I35
Mulling, 1950, 21, 68
'947. 19, 643
6 Huelin, F. E., & Stephens, I. Nyee, ilust. J . ex@.
Biol. wed. Sci. , 1947, 25, 17
7 McKenzie, H. A. , 1. Coun. scz. industr. IZcs. Aust.,
1945, 18, 18r
* McKenzie, H. A,, J . TOY. Soc. N.S.W., 1947, 81, 147
B Evenden, W., & Marsh, G. L., Food Res. , 1948, 13,
10 Huelin, F. E., Food .Rcs., 1953, 18, 633
11 J oslyn, M. A., & Miller, J ., Food 12es., 1949, 14, 325
1 2 Freed, M., Brenner, S. , & Wodicka, V. O., Food
13 Riester, D. W., Braun, 0. G., & Pearce, W. E.,
Tech. Chamflaign, 1949, 3, 148
Food Ind. , 1945, 17, 742
Received 28 May, 1958
l4 Feaster, J . F., Tompkins, M. D., & Pearce, W. E.,
l5 Huelin, F. E., & Stephens, I . Myee, Aust. J . sci.
l6 Huelin, F. E., & Stephens, I. Myee, Aust . J . sci.
l 7 Curl, A. L., & Veldhuis, 31. K., Frui t Prod. J. , 1948,
J oslyn, M. X., & Marsh, G. L., I ndust r. Engng
Chem. (Industr. Edn ) , 1934, 26, 295
Blair, J . S. , Godar, E. M., Reinke, H. G., &
Marshall, J . R., Food Tech. Champaign, 195 j , 11,
2o Blair, J . S. , Godar, E. M., Masters, J . E., & Riester,
D. Mi., Food Res., 1952, 17, 235
21 Loeffler, H. J ., Industr. Engng Chem. ( I ndust r.
2e J oslyn, M. A., & Marsh, G. L., Industr. Engng
23 Natarajan, C. P., & Mackinney, G., Food Tech, ,
2 4 Bayes, A. L., Food Tech., Champai gn, 1950, 4, I jr
2 5 Lueck, R. H., & Brighton, K. W., I nt . Congr. on
Food Res. , 1949, 14, 2 j
Res., 1948, I, 58
Res. , 1948, 1, 50
27, 342
Ed%), 1941, 33, 1308
Chem. ( I ndust r. Ed n ) , 1935, 27, 186
Chawflaign, 1949, 3, 373
Canned Foods (Paris), 19j1, 2, XIV-8
1.-The Quantitative Analysis of Phenolic Constituents
Methods for the quantitative analysis of anthocyanins, leuco-anthocyanins, flavanols
and total phenols in plant tissue extracts are critically examined and suitable modifications
of existing methods are described.
* Present address : Division of Forest Products, C.S.I.K.O., Melbourne, -1ustralia
J. Sci. Food Agric., 10, January, 1959
It has recently been suggested that the formation of the troublesome defect of gum in the
flesh (mesocarp) of canned Victoria plums may be related to the transport of phenolic glycosides
from the kernel and hence to the degree and rapidity of lignification of the stone (endocarp).l
Lignification in plants has been shown to be closely associated with the occurrence of leuco-
anthocyanins in leaves2 and it has also been suggested that these compounds are precursors of
heartwood extractives. An investigation of the variability in the amounts of leuco-anthocyanins
and other phenolic compounds in the leaves and other tissues of the Victoria plum (Prunus
domestica var. Victoria) during the course of a season was therefore undertaken to see what effect
these had on stone formation. Since the investigation involved the quantitative analysis of
over two hundred samples, methods involving preliminary separation of individual compounds
in each extract before analysis4 were out of the question. Methods in use in these labora-
tories for the analysis of individual groups of phenolic compounds were therefore modified and
improved and are reported here. The results of these analyscs will be given in Part 11.
Methods of Analysis.5
with n-butanol to 500 ml.
concentrated sulphuric acid.
g ml. of methanolic hydrochloric acid ( 5 : I v/v, 3 ~ ) .
Folin-Denis.-The reagents were prepared according to the procedure given in the A.O.A.C.
Leuco-anthocyanin reagent.--Concentrated hydrochloric acid (25 ml. of 3696 wiw) is diluted
Vani l h reagent.-One g. of recrystallised vanillin is dissolved in roo ml. of 707; (VJ 'V)
Anthocyanin reagent.-A freshly prepared solution of I ml. of 30% hydrogen peroxide in
The reagent should be prepared freshly every 3 days.
Total phenols.-A suitable aliquot of the solution under test, containing not more than
0.5 ml. of methanol or ethanol, is diluted with water to about 7 ml. in a 10-ml graduated test-
tube. The contents are well mixed, 0.50 ml. of the Folin-Denis reagent is added, and the
tubes are thoroughly shaken again. Exactly 3 min. later, 1.0 ml. of saturated sodium carbonate
solution is added and the mixture made up to 10 ml. with good mixing. After I h., the
absorptivity is determined in I-cm. cells at 725 m,u using as a blank water and reagents only.
If the solution is cloudy or a precipitate forms, it should be filtered or centrifuged before readings
are taken.
Leuco-anthocyanins.-One ml. of the solution under test (which should not be more than
50% with respect to either methanol or ethanol) is placed in each of two 6 x I in. ground-glass
stoppered test-tubes of uniform bore and wall thickness and 10.0 nil. of leuco-anthocyanin reagent
added. The tubes are shaken until the solution is homogeneous and one of them is placed
unstoppered in a constant-level all-metal water-bath maintained at 97 & IO. After 3 min.
the stopper is placed firmly in position and the tube heated for a total of 40 min. After removal
of the stopper, the tube is cooled in running tap water for j nun. and the absorptivit~of the
solution determined in a I-cm. cell at 550 m,u and, if chlorophvll is present, at 650 mp, using
as a blank the contents of thc unheated tube.
Flavanols.--A suitable aliquot of solution, containing not more than 0.1 ml. of methanol,
is placed in each of two 25-1111. conical flasks (A and B) and diluted to 2-0 ml. with water.
Four ml. of the vanillin reagent are added from a burette during 10-15 sec. to flask A, and
4.0 ml. of 70% (v/v) sulphuric acid to flask B, the flasks being shaken in a bath of cold water
to prevent the temperature from rising above 35". After shaking well the flasks are left for
exactly 15 min. at room temperature and the absorptivities of the contents of the two flasks
and that of a previously prepared blank (C) (4.0 ml. of reagent and 2.0 ml. of water) are measured
in I-cm. cells at 500 mp against 47y0 sulphuric acid (D) (4.0 ml. of 70% v/v H,SO, and 2.0 ml.
of water).
J. Sci. Food Agric., 10, January, 1959
The absorptivities of the reagent blank (C) and of the second flask (B) are subtracted from
the reading given by (A). Alternatively the cells may be arranged in the holder so that A + D
are read against B + C.
Anthocyanins.-Three ml. of hydrochloric acid in aqueous methanol (HC10.5~ ; CH,OH
80-85y0 v/v) are placed in each of two tubes. The solution in the first tube is diluted with
1.0 ml. of methanolic hydrochloric acid (5 : I v/v, 3N) , and 1-0 ml. of the peroxide reagent is
added to the second tube. The tubes are left for 15 min. in the dark and the absorptivity of
the solution in the second tube measured in a I-cm. cell at 525 mp using the contents of the
first tube as a blank. In all cases, standard curves were prepared using known amounts of
suitable compounds.
Results and discussion
Total phenols
The quantitative estimation of total phenols in biological extracts can be accomplished
in a number of different ways. Most phenolic compounds are readily attacked by various
oxidising agents ; many react with diazotised amines and, like electrophilic substances, form
coloured compounds ; some form coloured complexes with certain metals ; all show definite
absorption peaks in the ultra-violet, and methods for their estimation have been devised based on
such properties. Since, however, individual compounds vary widely in their ability to react with
(a) oxidising agents such as phosphomolybdate or permanganate,G ( b) coupling reagents such as
diazotised 9-nitroaniline' or Gibbs reagent,s (c) metals such as iron,s and (a) in their ultra-
violet or visible adsorption spectra ;lo all the procedures for the estimation of total phenols are
necessarily empirical.
I n the course of these invesigations it was found that methods based on the use of oxidising
agents are the most useful, since the variation between estimations of individual compounds
is much less than that obtained using methods based on the other properties listed above.
J oslyn and his co-workersG have shown that there is little difference in the results obtained with
natural extracts using either the modified method of Folin & Denis or that of Lowenthal, but
in our experience the former has proved to be more convenient.
A number of variations of the method have been examined ( eg 11) but none seemed to
offer any particular advantage. The addition of extra saturated sodium carbonate to the
solutions (2.0 ml. instead of 1.0 ml.), although increasing the likelihood of precipitation, was
found to make no difference to the reading obtained, but the use of other bases such as potassium
carbonate, sodium hydroxide or sodium phosphate buffers of various pH gave less reproducible
results The transmission curve of the blue complex showed a very broad maximum between
620 mp and 740 mp with a peak at 725 mp (cf. 12) and hence simple colorimeters can be used
with a suitable red filter without much loss of precision. All the phenolic compounds tested
showed an almost linear relationship between absorptivity and concentration, although the
slopes of the curves were different, the useful range using I-cm. cells being of the order of
10-100 pg. of substance in the aliquot taken for examination.
All quantitative estimations of leuco-anthocyanins are based on the transformation of these
substances to anthocyanidins by heating in acid solution. The transformation is not quanti-
tative since a large amount of brown ' phlobaphene-like ' polymer is formed along with the
anthocyanidin.13 The proportion of anthocyanidin produced depends on the solvent used ;
in aqueous acid, for example, not more than 10% is obtained, whereas in alcoholic solvents
the yield is of the order of 25%.
The first method devised for the estimation of leuco-anthocyanins involved purification
by multiple transfers between solvents of the anthocyanidin produced1* and is unsuitable for
routine analysis. A much simpler method using a direct measure of the colour produced on
heating the sample in isopropanol-HC1 at IOOO was introduced by Pigman and his co-workers,l5
but this suffers from the disadvantage that precautions must be taken to prevent evaporation
of the low-boiling solvent.
J. Sci. Food Agric., 10, January, 1959
A number of other solvents with a boiling point over 100' were thercfore investigated,
$2-butanol being found to be the most satisfactory. The colour development was more or less
complete in 30-3j-min. (cf. 15) and the Amax for leuco-cyanidins was found to be at 550 mp
(Fig. I). I n other solvents (water, acetic acid) the smaller shoulder at 450 mp developed into
a definite peak with a lowering of absorption at 550 mp (Fig. I) and this is undoubtedly due to
the relatively greater production of phlobaphene-like polymers. (+) -Catechin after heating
in m-butanol-HC1 gives a product having a similar peak at 450 mp (cf. 16).
..I J3oilrd in n-butanol HCI
B Boiled in acetic acid-HCl
C (r)~Cat~chi n boiled in n-butanol-HC1
Using the method described or that of Pigman et al.,15 good replicates can only be obtained
when careful attention is paid to obtaining uniform heating conditions. This necessitates
the selection of tubes of uniform bore and wall thickness, maintenance of a constant level of
water in the heating bath and good control of temperature. The anthocyanidin produced
fades very slowly in the dark, but this process is accelerated by light1: and therefore a metal
bath constructed so that no direct light falls on the tubes, should be used for the analysis.
When methanolic leaf extracts were analysed for leuco-anthocyanins by the method
described, i t was observed that chlorophyll contributes to the absorption at 550 mp. The
visible spectrum of chlorophyll-containing extracts free from Zeuco-anthocyanin (e.g. from
nettle leaves) was, however, found to be unchanged after heating in the butanol-HC1 reagent.
This observation was checked in routine determinations by measurement at 650 inp where
chlorophyll absorbs strongly and anthocyanidins do not intcrferc, and indicated that an un-
heated control can be used as a blank.
Similarly the contribution to the adsorption at 550 mp made by anthocyanins in extracts
from fruit skins was found to be unchanged after heating in the reagent, although in this case
hydrolysis of the glycosides undoubtedly occurs. It is apparent that the molecular extinction
coefficient of anthocyanidin glycosides is little different from that of aglycone and in this case
also an unheated control can be used as a blank. The useful range of the method using I-cm.
cells is 50-400 pg. but an absolute straight-line relationship between concentration and absorp-
tivity was not obtained.
The colour reaction for the detection of phloroglucinol using vanillin and concentrated
hydrochloric acidla has been used both q~al i tati vel yl ~ and quantitativelyz0>21 for compounds
other than phloroglucinol which contain the I : 3 : 5-trihydroxybenzene nucleus. The red
colour produced is due to the formation of a carbonium ionz2$ 23 which is unstable even in the
presence of 13%) hydrochloric acid. 22 Attempts to apply the Lindt reagent quantitatively
were found to give non-reproducible results, which was apparently due to the instability of
the carbonium ion form, since the difficulty could be overcome by using 7096 sulphuric acid
instead of 30% alcholic hydrochloric acidz1 in making up the reagent. Under these conditions
the adduct with both phloroglucinol and catechin had A,,,, 500 m,u (cf. 23) and the colour formed
only faded slowly (Fig. 2) . Even so i t can be seen that care must be taken to measure the absorp-
tion at a fixed time after addition of the reagent. I t was found essential for best rcproducibility
that the initial mixing of the reagent with the solution under test should be done as rapidly as
possible and that the temperature of the solution should be kept below 40'.
The presence of excessive amounts of methanol, or more especially ethanol, materially
J, Sci. Food Agric., 10, January, 1959
alters the colour obtained in the reaction to a bluer shade. This is probably due to the formation
of a coniferylaldehyde-like compound by the vanillin and alcohol in strong sulphuric acid which
then condenses with the phenol. Such addition products are known to have absorption maxima
at longer wavelengths than the original compound.z2
TIME, min.
FIG. ?-.-Dewease of optical density of vanillin-flavanol adduct with time
Examination of the method showed that only compounds containing an undeactivated
phloroglucinol (or resorcinol) nucleus react when present in amounts in the useful range of the
method (0-100 pg. in the aliquot). Compounds such as phloracetophenone, butein, hesperidin
or quercetin gave no colour with the reagent. Measurements showed that Beer's law was obeyed
for all reactive compounds tested.
A nthocyanins
The qualitative estimation of anthocyanins in aqueous buffer extracts of fruit tissues can
be satisfactorily carried out by measuring the difference in the red colour of the extract at
pH 2-0 and pH 3.4.24 As can be seen from Fig. 3, however, the lower pH recommended by
the American workers is not low enough to ensure highest sensitivity or accuracy since at this
pH the change in colour with a small change in pH is still large (cf. 25) . It was found better
to use a pH between 0.5 and 1.0 (Fig. 3) . The change in absorptivity in the visible spectrum
of the anthocyanin solution with pH is dependent on the change in the proportion of the com-
pound in the oxonium-carbonium ion form,26 and it is interesting to note that the absorptivity
due to the phenolic hydroxyl groups at 280 m,u is not changed greatly by changes in pH from
I to 5 (Fig. 3) .
OO I 2 3 4
When methanolic hydrochloric acid was used to extract the tissue, however, the above
method was found to be unsatisfactory because of the large dilution required to ensure adequate
buffering. The possibility of bleaching the solution without change in pH was therefore
examined. A recommended methodz5 using sodium sulphite was found to require far more
of the reagent than had been suggested, causing cloudiness, and hydrogen peroxidez7 was
therefore substituted. Under the conditions used, decolorisation of a standard anthocyanin
solution was found to be virtually complete in j min. and the reagent showed no serious influence
on the colour of other substances (such as chlorophyll) present in extracts from immature fruit
J. Sci. Food Agric., 10, January, 1959
The method was found to be suitable over a range of 0.30 pg. of anthocyanin and the curve
(Cyanin hydrochloride, 10 pg.,/ml., in methanolic HC1 obtained was linear with concentration.
gives optical density of 0.405 in a I-cm. cell at 525 mp.)
The work described in this paper was carried out as part of the programme of the Depart-
ment of Scientific and Industrial Research and of the Division of Forest Products, Commonwealth
Scientific and Research Organisation, Australia.
The authors wish to thank Dr. W. G. E. Forsyth for a gift of pure cacao Zeuco-anthocyanin.
Crown Co$yi ght Reserved
Low Temperature Kesearch Station
Downing St.
1 Bate-Smith, E. C., k Pridham, J . B., Personal
Date-Smith, E. C., Biochoin. J. , 1954, 56, 1 2 1
Hillis, W. E., Aust. J . biol. Sci., 1956, 9, 263
'ridham, J . B., Anal y t . Chem., 1957, 29, 116j
' Official and Tentative J Iethods of -1nalysis ',
Ass. Off. hgric. Chem., Igjj, 8th Edn, p. 144
(Washington : A.O.A.C.)
Smit, C. J . B., J oslyn, 31. h. , & Lukton, A,
Anal yt . Chem. , 19jj. 27, 11j9
' Bray, H. G., Humphris, 3. G., Thorpe, W. V.,
White, K., and Wood, I?. B., Bi ocham. J. , 19.52,
* King, I.. E., King, T. J ., c% Nanniiig, L. C., J . chem.
52, 4.16
lo -4ulin-Erdtrnai1, G., Sz,r7is/z. Pappe~st i di zi 7zg, 19j 3,
56, 287
I 1 Englis, I>. T., Miles, J . \Ir., & Wollerman, 1,. -I.,
J . As s . off. agvic. Cheiu., Was h. , 1955, 38, 51s
l9 Pro, 31. J .. J . Ass. 04.. a p i r . Cheiiz., Wash. , 1952, 35,
XI Bate-Smith, E. C., S: Slrain, T., Cizeix. G. Ii zd. ,
7953. P. 377
Received 14 March. 1958 ; amcndfrl iiianuscript 25 J uly, ryj S
l4 I-Iallas, C. A., Keseavch Hecovd h'o. 70, 1939, Brit.
Ass. Res. for the Cacao, Chocolate, Sugar Con-
fectionery and J am Trades (quoted by Forsyth,
W. G. C., Biochem. J. , 1952, 51, 516)
l5 Pigman, W., Anderson, E., Fischer, R., Buchanan,
M. A, , & Browning, B. L., TAPPI , 1953, 36, 4
l 6 Hathway, D. E., & Seakins, J . W. T., J . chem. Soc.,
1957. P. 1562
l 7 Nordstrom, C. G., Ac t a chem. scand., 1956, 10, 1491
l9 Bradfield, A. E., & Penney, I T., J . chem. Soc., 1948,
Lindt, O., Z. anal. Chem. , 1887, 26, 260
P. 2249
Kursanov, A. L., Biokhimiya, 1941, 6, 128
21 W-atkin, J . E., Underhill, E. W., & Neish, A. C.,
2 2 Pew, J . C., J . Amev. chenz. Sor.. Iq-jI, 73, 1678
2 3 Giniliano, R., Ann. chi m. apfil., Kortza, 1939, 29, 86
24 Sondheimer. E.. & Iiertesz. Z. 1.. Aimlvt. Chent..
Cnnad. J . Bi ochem. , 19.57, 35, 229
Soc , 1940, 62, 2711
27 Karrer, P., & de Xleuron, G , Helv chwn Act a, 1932,
15, 507
Using seven different methods of preparation, the sedimentation, tenacity, in vitro
fungitoxicity, and rate of crystallisation of 8 : 10 : 100 Bordeaux mixture have been in-
vestigated. It is suggested that the most effective Bordeaux mixture is that prepared by
adding a concentrated lime paste to diluted copper wlphate.
Although long-established as a fungicide, Bordeaux mixture is still widely used in plant
protection. It has been demonstratedl that the chemical properties of the gelatinous precipitate,
formed by copper sulphate and calcium hydroxide, are not dependent on the manner in which
J. Sci. Food Agric., 10, January, 1959