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The microbiology of instant tea
Food Microbiology, 1987, 4, 19-33
The microbiology
of instant tea
V. Vanes*, S. Hofstaetter
and L. Cox
Nest16 Products Technical Assistance Company, Central Quality Assurance Laboratory, Au. Nestle’, 55 CHl800 Vevey, Switzerland Received
15 October 1986
The microbial ecology of an instant tea line was studied using samples from different points of the line. Only lactic acid bacteria were found at certain manufacturing stages. The main contaminant was identified as Lactobacillus plantarum. As no other bacteria were found in this line, lactic acid bacteria may be considered as quality ‘indicators’ and their determination might be a means to monitor Good Manufacturing Practices (GMP). In addition, the total tea solids (TS) concentration that inhibits the growth of certain isolated L. plantarum strains was determined on the instant tea line. A TS concentration of 60% inhibited these organisms during48 h. The same TS concentration did not inhibit the growth of a non-identified cluster of Lactobacilli isolated from the same line. The bacteriostatic effects ofpure theophylline andpure theobromine on thegrowth of L. plantarum were also examined. No bacteriostatic effect in the presence of either compound was observed at normal concentration or at concentrations higher than those calculated to be normally present in tea lines. The standard aroma profile of an instant tea solution was modified as a result of inoculation with a wild strain of L. plantarum. This implies that heavy contamination and fermentation by such organisms might result in products with altered aroma profiles that are unacceptable to the consumer. It should be noted that altered tea aroma might eventually be used as an indicator of microbial build-up by analysing certain of the altered aroma components. Further work is nee&d to investigate this possibility. Challenge tests with known pathogens and spoilage organisms revealed that, although pathogens were unable to multiply in instant tea solutions, spoilage organisms could grow or survive and thus might pose problems.
Introduction Despite the wide consumption of instant tea, its microbiology is so far practically unknown. The objective of our study was to detect and identify the bacteria present at various points of a tea line and, according to the results thus obtained, to define quality ‘indicator’ * Present address: Nestle products Technical Assistance Company, Central Quality Assurance Laboratory, Av. Nestle, 55 CH1800 Vevey, Switzerland. (To whom correspondence should be addressed.) 0740-0020/87/010019
+ 15 $03.00/0
micro-organisms which might be used to monitor the hygiene aspects of Good Manufacturing Practices (GMP) in an instant tea factory. It is well known that tea contains elements such as alkaloids (the meth ylated derivatives of purine: caffeine, theobromine and theophylline) and catechins (flavonols), which may have a bacteriostatic effect (Lunder 1979). Caffeine levels vary between 3 and 5% in tea leaves and in manufactured tea (Lunder 1979) and are at about 4% in fresh tea shoot tips (Rehm and Reed 1983). 0 1987 Academic
Press Inc. (London)
Limited
20
V. Vanos et al.
Theophylline is present in the range of 0.23 to 0.44 mg per 100 g, and the theobromine content is approx. 50 mg per 100 gin manufactured tea. Flavonols (mostly catechins) constitute about 20% of the dry weight of tea leaves (Lunder 1979). Tea infusions are inhibitory to microorganisms, having a bacteriostatic effect in vitro on Salmonella typhi, Shigella paradysenteriae, Shigella dysenteriae, Staphylococcus aureus, Vibrio cholera and Leuconostoc mesenteroides (Lunder 1979). Little is known with regard to the effect of the concentration of natural inhibitors in tea on bacteria in general, especially those found in instant tea or potential spoilage and lines, pathogenic micro-organisms. For this reason, an attempt has been made to determine the tea constituents with the most inhibitory effects and to find out at what TS concentrations the tea solution completely inhibits growth of the predominant microflora. Among the alkaloids, the bacteriostatic effect of caffeine on certain bacteria was discussed in a previous article (Vanes and Bindschedler 1985). In the present study, the bacteriostatic effects of pure theophylline and theobromine have been examined as well as those of catechins as part of the examined tannin complex. The growth kinetics of some pathogenic and non-pathogenic bacteria in reconstituted soluble tea have also been studied. In this context potential
Table
1. Colonies
taken
from
different
hazards related to tea consumption have been considered. The modification of the standard aroma profile by controlled fermentation was also investigated.
Materials and Methods Microbiological profile of instant tea Ten samples from different points of a tea line were examined in duplicate (see Table 1, where only the points of the tea line which had presence of bacterial flora, are mentioned) using Chalmers’ medium (Vanos and Cox 1986) modified at our laboratory. Two well-dried plates per dilution were inoculated by surface spreading. One of the plates was covered with 8 ml of 15% water agar to obtain better ‘micro-aerophilic’ conditions, which are necessary for the detection of certain lactic acid bacteria. Plates were then incubated at 30°C for 72 h. In order to classify the isolated bacteria into groups, all colonies were first purified twice at 30°C on Plate Count Agar (PCA) OXOID CM 325 containing 1.5% sterile skim milk OXOID L31 (10% reconstituted). Afterwards, the following tests were carried out: 1. Cat&se test: Reaction with 30% H202 to detect the presence of the enzyme catalase. 2. Gram staining: Observation of the cell morphology and the possible presence of spores. 3. Growth tests in MRS broth at diferent temperatures: 10,30,37 and 45°C during 48 h of incubation. 4. Homo-heterofermentation test: To detect strains producing gasfrom glucosefermentation (Mossel and Tamminga 1980). 5. A.DJI. test: To detect the presenceof the arginine dihydrolase enzyme in the isolated strains (Institut Pasteur 1981). 6. Ability to ferment ribose(FLU&I 83860).
points
in the tea-line.
No. of sample Point of the tea-line 1
2 3 ii
Concentrated liquor after evaporation Distillate Clarified liquor in surge tank Concentrated liquor in surgetank Spray-mix in HP pump surgetank
Coloniestaken 66 5: 20 67
Microbiology To identify the Lactobacillus species, the following substrates were used: From MERCK: D-Arabinose (1494); L-Arabinose (1492); Dulcitol(5990); meso-Erythritol (3160); Esculin (842); D(+) Glucose (8342); meso-Inositol(4728); Lactose (7657); Maltose (5910); D-Mannitol (5982); D(+) Mannose (5984); Raffinose (7549); Salicin (7665); L(+) Rhamnose(4736); D-Xylose (8692). From FLUKA: D( +) Galactose (48260); Gentiobiose (Amygdalose) (48790); Fructose (47740); D(+) Melibiose (63630); D(+) Saccharose(84100); DSorbitol(5530). The ability of the isolated strains to ferment these substrates was tested in MRS broth in which glucosewas replaced by the respective substrate at a concentration of 2%, with 0.001% bromocresolpurple as pH indicator. Becausecertain sugar fermentations are in general plasmid-linked (Gasson and Davies 1984), a positive identification was recorded if 20 out of the 21 substrates gave typical results, offering a confidence limit of 95%. Furthermore, the configuration of the lactic acid was examined by optical rotation, asa complementary test to identify certain strains. The API 20 STREP identification kit was usedto identify the Streptococcus strains. Strains used Lactobacillusplantarum Lactobacillus sp.
(cluster F) Streptococcus fwcium Pediococcus pentosaceus Bacillus cer&s Escherichia coli Salmonella typhimurium Staphylococcus aureus Streptococcus faecalk Saccharomyces cerevisiae
Culture
of instant tea
21
Media For the purification of the non-lactic strains, the following media were used: B.cereus: S. aureus: S. faecalis: E. coli: Salmonella
typhimurium: Saccharomyces cerevisiae:
M.Y. agar (MERCK 5267) + supplements (egg yolk + polymyxin) Baird Parker (OXOID CM 275) + supplements (eggyolk + tellurite) KF agar (DIFCO 0496-01-o V.R.B.L. agar (DIFCO 0012-01-05) B.G.A. (OXOID CM 329) O.G.Y.E. agar + supplement(OXOID CM 545)
For plate count determinations, the following media and incubation conditions were applied. Lactic strains: MRS agar, (OXOID CM 361); Incubation at 30°C for 72 h. Non-lactic strains: Plate Count Agar (PCA, OXOID CM 325) + 1% triphenyl tetrazolium chloride (‘ITC) from a 1% solution (DIFCO 3112-67-9) was used for all strains except Origins
QA laboratory, isolated from tea line QA laboratory, isolated from tea line QA laboratory, isolated from tea line QA laboratorv. isolated from tea line ” Lausanne2868 ATCC 25922 ATCC 14028 ATCC 25923 ATCC 206 Linor (NESTEC)
conditions
The non-lactic strains were first purified on selective media before being cultured in Trypticase Soy Broth (TSB BBL 11043) at 37°C. Lactic strains were cultured in MRS broth at 30°C. To obtain cultures with maximum activity, each strain was subcultured three times in the corresponding broth before use. For the experiments, all strains were taken at the late log phase.
culture collections
B. cereus and S. aureus where the respective
selective media were used. Incubation: 37°C for 24 h. Dilutions were made in tryptone-salt (l-8.5 g-1). Inoculation techniques for solid media Double layer (overlay the poured inoculated layer with the same medium) for MRS medium; poured plates for the PCA medium and surface spreading for M.Y. and BairdParker media.
22
6. Vanos et al.
Preparation of soluble tea For the preparation of a soluble tea solution, 2 g of instant tea powder were added to 100 ml of distilled water at 100°C; after 10 mins the tea was cooled to 37°C. This method resulted in less than 10 colony forming units (cfu) per ml.
sterile 2% TS tea solutions. In 10 and 12% solutions the pH was 7.7. The growth of L. plantarum was examined in these solutions. The same assay was repeated by adjusting the pH to 6 in the 10 and 12% tannin solutions. Growth of pathogenic and spoilage micro-organisms The solutions of instant tea (2%) were inoculated with the different micro-organismsat a level of 104-107 cfu ml-i. They were incubated at 30°C in all solutions during 48 h. At intervals of 4, 8, 16, 24 and 48 h they were plated in the corresponding selective media.
Bacteriostatic effect of theophylline Despite its low concentration, theophylline could contribute synergistically to the bacteriostatic effect of instant tea. Sterile pure theophylline (FLUKA 88308) was added to a final concentration of 0.3 mg ml-l to 2% TS instant tea solution. This theophylline concentration is about 200 times higher than in a normal instant tea Preparation and fermentation of soluble tea solution with a TS content of 60%. The solution was inoculated with L. plan- The tea powder was reconstituted at 2% in tarum (lOa--lOa cfu ml-i) and incubated at sterile distilled, boiling water in four screw30°C during 48 h. At intervals of 4,8, 16, 24 cappedbottles and then cooledto 37°C. Two of and 48 h, L. plantarum colonieswere counted them were inoculated with the same inocuin the corresponding selective medium. lum from the L. plantarum strain isolated from the tea line to produce 10-10s cfu ml-i. These bottles, with one control bottle, were Bacteriostatic effect of theobromine incubated at 30°C for 48 h. The other control Despite its low concentration, theobromine bottle was analysed immediately for the could also contribute synergistically to the aroma profile. bacteriostatic effect of instant tea. Since the pure theobromine used (FLUKA Screening of the aroma profile of 88304) was not water-soluble, a solution of fermented soluble tea 5% was made in dimethylsulfoxide (DMSO) The analyse the aromas of fermented tea, a (MERCK 802912). DMSO did not inhibit gas chromatograph HP5880 was used with a lactic acid bacteria. This solution was then headscapetrapping apparatus (J. Rectorit), added to 2% TS instant tea with a final (trap 6 min, N2 = 60 ml min-1, GC: 15 min concentration of 0.3 mg ml-i, the normal 180°C(2°C min-i), P = 1 bar, split = 10 ml concentration of 60% TS instant tea. The min-1). The analysis was performed in duplisolution was inoculated with the samestrain cate for fermented and non-fermented soluble of L. plantarum and the above procedure was tea. repeated. Water activity (ad measurement Bacteriostatic effect of catechins The a, was measured with a NOVASINA The tannins found in the tea leaves are not a EEJA-3 apparatus. Calibration for the 0.902 single chemical compound but rather a mix- value was made at 25°C by using saturated ture of polyphenols representing as much as BaC12. 30% of the dry weight of the leaves (Lunder 1979). pH measurements In this mixture of polyphenols, flavonols (known as catechins) represent about 83% The pH was measured with a digital pH(Lunder 1979). Since the catechin complex Meter (E 532 Metrohm Herisau). was not commercially available, its bacteriostatic effect on Lactobacillus growth was examined indirectly by using a 12% tannin Results solution which was obtained from the factory as the maximum concentration of tannins A total of 212 colonies were picked at found in the tea line. Dilutions of this random (picking the square root of the solution were made at 2, 4, 5, 8, and 10% in number of the colonies of the examined
V = variable.
-ve
-ve
Pediococcus
Atypieals
Streptococcus
-ve
Streptobacteria (Rogosa’s classification)
Lactobacillaceae
Streptococcaceae
-ve
Group
Catalase
of the groups
Family
1
Table 2. Characteristics
+ve non-spore forming rods +ve cocci +ve cocci in tetrads
+ve non-spore forming rods
Gram staining
of bacteria
-
+
+
f
10
+
+
+
f
+
4
+
+
37
-
+
V 50% of the strains were +ve -
45
from the tea-line Growth at (“C)
30
isolated
-ve
-ve
--Ye
-ve
Gas production from glucose
+ve
+ve
-ve
-ve
of arginine dihydrolase
A.D.H. presence
as well as the number
-ve
-ve
-ve
+ve
fermentation
Ribose
of isolated
iii
4
9
30
169
isolated strains
Number of
strains.
24
V. Vanos et al.
plates)
from the plates for identification.
Table 3 shows the substrate utilization profiles of the identified and unidentified strains. Table 4 summarizes the different points of the tea line, from which the identified species were isolated. The results of growth studies at different TS concentrations are presented in Figures 1, 2, 3, 4 for each species of isolated bacteria (L. plantarum, Lacto-
Apart from some samples which did not carry any bacteria, only lactic acid bacteria were detected in the positive samples. Table 1 summarizes the numbers of bacteria isolated from the various samples as well as the number of colonies taken for identification. Table 2 lists some characteristics of the groups of bacteria isolated from the tea line, as well as the number of strains isolated per group.
Table 3. Use of substrates
by the strains
Clusters of Lactobacillus Substrates
bacilli-cluster saceus) .
F, S. faecium,
The growth of the dominant
isolated
Cluster of Streptococcus strains (number of strains)
Cluster of Pediococcus strains (number of strains) A (41
A(1201
B(30)
c(10)
D(1)
E (1)
F (281
A (8)
Amygdalose Arabinose D Arabinose L Dulcitol Erythritol Esculine Fructose Galactose Glucose Inositol Lactose Maltose Mannitol Mannose Melibiose Raffinose Rhamnose Salicine Sorbitol Sucrose Xylose D
+
+
+
+ + + + + + + + + + + + + + -
+ + + + + + + + + + + + + + + + -
+ + + + + + + + + + + + + + + + -
+ -
+ + + + + + + + + + + + + + +
+ + + + + + + + + + + + + + + -
+ +
Lactic acid configuration
DL
Identified species
L.plantlVU?Tl
Atypical but very closeto L. plantarum
organism,
from the tea-line.
strains (number of strains)
Not examined
P. pento-
+ + + +
-
+ + + + + + + + + +
Not Not Not examined examined examined Atypicals
S. faecium
P. pentosaceus
Microbiology Table 4. Percentage
of species isolated
from different
of instant
Isolated species (o/oof total isolated bacteria)
Concentrated
L. plantarum (84) Atypical lactobacilli (16) P. pentosacew (80) S. faecium (20) L.plantarum (40) Atypical lactobacilli (20) S. faecium (40) L. plantarum (61) Atypical lactobacilli (39)
Distillate Clarified liquor in surge tank Spray mix in HP pump surge tank
L. planturum, is shown in Fig. 1. Inhibition began at 10% TS and was complete at 60% Ts. The growth of the Lactobacilli-cluster F is shown in Fig. 2. Inhibition was not complete, even at 60% TS, but growth was quite slow at this. These bacteria grew better at 10, 20 and 30% TS than at 2% TS. The growth of S. fuecium was completely inhibited at 30% TS (Fig. 3). This strain grew best at 10% TS, whereas at 2% TS a rapid die-off occurred after 16 h incubation. The growth of P. pentosuceus was inhibited even at 2% TS (Fig. 4). This strain was a mesophile and did not grow at 45°C after 48 h incubation.
The growth dynamics of the dominant species L. plantarum in the presence of pure theophylline and theobromine are presented in Figs 5 and 6. No inhibition occurred at 0.3 mg ml-l during 48 h with either compound. The bacteriostatic effects of different tannin concentrations on the growth of the dominant strain L. phturum are shown in Figs 7 and 8. At 10 and 12% of tannins, growth was inhibited during 24 h at pH 7.7 but not at pH 6. The growth kinetics of E. coli and S. typhimurium are presented in Figs 9 and 10. After incubation at 37°C for 48 h, E. coli could not be detected. Salmonellae
TS % * --__-,o . . . . . . . . . zoso------
I
60----I 4
I 8
I 16
I 24
I 48 Tome
Fig. 1. Growth
of L. plantarum
25
points of the tea-line.
Point of the tea-line liquor after evaporation
tea
(h)
in instant tea at different TS concentrations.
-
-
26
V. Vanos et al. TS %
I
j
I I
I
I
4
8
I 6,
48
24 Ttme
Fig. 2. Growth
60-
I
I
of cluster F of lactobacilli
(hi
in instant tea at different TS concentrations.
TS %
8 7
:
t
.,..........
. .._...................
...
2
. . .. . . .. ..
---__
I
Tome (h)
+ Fig. 3. Growth
of S. foe&m
in instant tea at different TS concentrations.
TS % 8-
73 ‘;
6-
/ /
5-
1
2
-----
IO ’ 20 30 40 50
1
1 4
I 8
I I6
1 24
I 48 7me
Fig. 4. Growth
r--1
ih i
of P. pentosaceus in instant tea at different TS concentrations.
Microbiology
Fig. 5. Growth of L. plantarum in instant tea at 2% TS (- - -) and in instant tea at 2% TS containing O-3 mg ml-1 of theophylline G--h
‘r
4
8
16
24 Time
of instant
tea
27
decreased rapidly after 24 h and were not detected after 48 h. Under the same conditions, S. aureus was not detected either (Fig. 111, whereas the number of S. faecalis remained constant during 48 h (Fig. 12). B. cereus colonies were not detected even after 16 h (Fig. 131, whereas the number of S. cereuisiae remained constant during 48 h of incubation (Fig. 14). The instant tea solution, inoculated with L. planturum and incubated at 30°C for 48 h, clearly showed qualitative changes in the aroma profile (Figs 15, 16). Unidentified new peaks with retention times (R,) of 30 and 68 min were detected in duplicate samples, and the peak with Rt 20 min had decreased. The sterile instant tea solution, incubated at 30°C for 48 h, did not show any modification of the standard aroma profile in comparison with the non-incubated sterile instant tea solution (Figs 17, 18).
48 ( hi
Fig. 6. Growth of L. pluntarum in instant tea at 2% (----) and in instant tea at 2% containing O-3 mg ml-l of theobromine G--1.
Discussion In the examined samples taken from different points of the tea line, the predominant microflora was found to be entirely constituted by lactic acid bac-
*.----
4-
-
-
6---
8--IO-
Fig. 7. Growth of L. plantarum in instant tea (2% TS) at different concentrations 10 and 12%, the pH was found to be 7.7.
-
-
of tannins. At
28
V. Vanos et al.
Fig. 8. Growth of L. plantarum
in instant tea (2% TS) at different concentrations of tannins. The pH was adjusted to 6 at 10 and 12%.
) sterile distilled water, (- - - -1 instant tea
Fig. 9. Survival ofE. coli strain ATCC 25922. (2%.
$-:7:-i ,,,_,,_,,___,: 4
l
8
16
24 Time
Fig. 10. Survival of Salmon&z water, (- - - -) instant tea 2%.
typhimurium
teria, mainly of the genus Lactobacillus. L. plunturum was the prevalent identified species, probably because of its ability to tolerate a relatively high concentration of oxygen, compared to the other species (Gotz et al. 1984). The same
48
(h)
strain ATCC 14028. (-)
sterile distilled
species had also been predominant in an instant coffee line as observed in an earlier study (Vanos and Bindschedler 1985). Although the conditions for growth ofLactobacilli (temperature, acid pH, presence of vitamins, amino acids
Microbiology
y.
4
I6
8
l ‘.
of S. aureus
8
16
16
‘1
‘.
b48
8
I 48 (hi
sterile distilled water, (- - - --I instant
24 Time
(h)
Fig. 13. Survival of B. cereus strain Lausanne 2868. (-) instant tea 2%.
sterile distilled water, (- - - -)
7 l ; z
61 5r). 4<
,*
-0-e - -.----
..-+--
L----L-, 4
8
16
cerevisiue
____-_--
.---
24 Time
Fig. 14. Survival of Saccharomyces (- - - -1 instant tea 2%.
29
sterile distilled water, k----I
24
Fig. 12. Survival of S. fuecalis strain ATCC 206. (-) tea 2%.
8
‘1
_----------_
Time
4
‘\
tea
ih)
strain ATCC 25923. (-)
S----S
4
‘,
24 Time
Fig. 11. Survival instant tea 2%.
‘,
of instant
-0
1
48 (h)
strain ex Linor. (-
) sterile distilled water,
30
V. Vanos et al. NESTEA
CONTROL
SAMPLE
(WITHOUT
j
INCUBATION)
12.70
8.71
Fig. 15. Aroma profile of a sterile instant tea solution.
I
NESTEA
+ L PLANTARUM
IO*00
20~00
30.00
Top Raw: BTHEOZ Bottom Row: BTHE03
(O-O-80.0) (O-O-80.0)
(Enlarged (Enlarged
CPOO Relative
40.00
(INCUBATION
50.01
48
H)
60.01
70.01
Plots x 30.0) x 30.0)
Fig. 16. Aroma profile of an instant tea solution inoculated with L. plantarum.
NESTEA
CONTROL
SAMPLE
(WLTHOUT
INCUBATION)
3.06
8.76 7.69
Fig. 17. Aroma profile of a sterile instant tea solution.
80.1
Microbiology INCUBATED
12.70
- _
I I.70
-
IO-70
-
9.70
-
8.71 7.71
i 0.00
NESTEA
SAMPLE
(INCUBATION
48
of instant tea
31
H)
1Ii IO.00
20.00
Relative Plots Top Row: BTHEOI Bottom Row: BTHE02
(0.0-80-O) 10*0-80.0)
30.00
( Enlarged i Enlarged
40.00
50.01
60.01
70.01
SO4
x 30.0) x 30.0)
Fig. 18. Aroma profile of the same sterile instant tea solution incubated at 30°C for 48h. and sugars) are less restrictive in a tea line than in a coffee line, the diversity of the detected Lactobacilli species was not the same. Of the 212 isolated strains, 93% were identified as Lactobacilli, 4.5% as Streptococci and 2.5% as Pediococci (Table 2). In the Lactobacilli group, 60% were identified as L. plantarum and the rest as atypical (Table 3, cluster A). The atypical Lactobacilli were classified into clusters (Table 3, clusters B, C, D, E, and F) according to their ‘substrate use’ profiles. Clusters B, C, D, and E were very close to the L. plantarum substrate profile, differing in the use of only two or three substrates. Cluster F (28 of the strains) differed considerably from the L. plantarum substrate profile. These strains did not ferment arabinose L, mannitol, melibiose, raffinose, sorbitol or sucrose, but only rhamnose. They were very similar to the species L. casei, Subsp. rhumnosus, but with a confidence limit below 90%. All the isolated Lactobacilli were mesophiles, of which 85% were classified as Streptobacteria as indicated in Table 2 (Rogosa and Sharpe 1959). All Lactobacilli grew at 10°C during 48 h incubation, and 50% of the Streptobacteria strains grew at 45°C. Therefore, to prevent multiplication of
Lactobacilli in the tea line, temperatures should be either higher than about 50°C or lower than 10°C. The absence of heterofermentative Lactobacillus species among the isolated strains might reduce the possibility of gas production as a problem in such a line, if abundant multiplication occurs during the weekend in insufficiently cleaned pipes. The presence of group D Streptococci, although they may survive in tea solutions, does not seem to represent a health hazard, due to their low incidence (4.5% of all isolated strains). The species present in this group was S. faecium. The examined strains could grow at 45°C. Other strains, however, can grow at 52°C. S. faecium was detected only in two samples from the distillate and from the concentrated liquor in the surge tank. As this strain had a very low incidence (4.5%) among the identified flora, it cannot be considered as a quality indicator of the tea line. Probably it does not originate from the line, but could be due to airborne post-contamination at the moment of sampling. There was a very low incidence of pediocoeci (only 2.5% of the total isolated flora) and therefore their presence would not seem to pose any problems. The growth of L. plantarum was com-
32
V. Vanos et al.
pletely inhibited in a tea solution of 60% TS. In experiments with 40-45% TS, inhibition of the growth persisted only for 16 h. This could mean that the growth of L. phtarum will not be completely inhibited under normal processing conditions in a tea line, where the maximum amount of TS in some parts of the line is 4M5%. Lactobacilli-cluster F tolerated the natural bacteriostatic elements of the tea, even at a high concentration. This group of Lactobacilli seems to represent a more important spoilage hazard for the tea line than L. plantarum. A P. pentosaceus strain was isolated from only one sample of the tea line and died off rapidly in instant tea solutions. It is possible that this species was not a true contaminant of the examined tea line, and that its presence in that particular sample resulted from accidental contamination during sampling. As mentioned before, L. plantarum was completely inhibited at 60% TS, at which concentration water activity was O-96 and pH 5-3. Neither of these values inhibits the growth of Lactobacilli (ICMSF, 1980). Therefore, the inhibition at 60% TS might have been caused by the high concentration of catechins and the alkloid complex ‘caffeine + theobromine + theophylline’. A bacteriostatic effect of caffeine on the growth of L. plan&rum has already been well demonstrated in a previous study (Vanos and Bindschedler 1985). In the present study, the alkaloids theobromine and theophylline have been investigated for their possible bacteriostatic effects on the dominant strain, L. plantarum. No bacteriostatic effect was observed with either compound, not even when theophylline was used at a concentration 200 times higher than normal. Therefore, the complex Ylavonols (cate-
chins) + caffeine’ seems to be the only natural inhibitor of Lactobacilli in instant tea. Complete inhibition of L. plantarum growth was obtained with a 12% tannin solution. The pH of that solution was 7.7 (Fig. 7). When pH was adjusted to 6.0, inhibition persisted only for 16 h (Fig. 8). Thus, the bacteriostatic effect of the tannin complex might be pH-dependent. However, at pH values higher than 7 Lactobacilli are inhibited (ICMSF 1980). A synergism between pH and tannin bacteriostasis is therefore difficult to confirm. The growth kinetics of pathogenic bacteria (Figs 9, 10, 11, 13) in instant tea solutions show that tea liquor is not a suitable medium for active growth of these strains, even if larger inocula are used than might occur naturally in a tea line. The Streptococci group D (S. faecium and S. fuecalis) and the yeast S. cereuisiae could represent a spoilage hazard for the tea line, since they are able to grow in this substrate. Lactic acid bacteria can be considered as ‘indicators’ of the quality in a tea-line, because no other bacteria such as coliforms, spore formers, etc were detected. However, the extent of their presence and its true significance remain to be investigated. The inability of certain pathogenic organisms to grow in instant tea solution indicates that public health hazards are most unlikely to arise in relation to this product. Moreover, further investigations into the modification of tea aroma by Lactobacilli are required before any final conclusion may be drawn regarding the significance of these micro-organisms for the sensory quality of instant tea.
Microbiology of instant tea 33 Acknowledgements We wish to thank Dr J. C. Spadone, Laboratory of Organic Chemistry, Central Research Department of NESTLE (Vets-ehez-les Blanc, Switzerland), for providing the aroma profiles of the fermented and non-fermented instant tea solutions.
References Buchanan, R. E. and Gibbons, N. E. (1974)Bergey’s manual ofdeterminative bacteriology, 8th edn. Baltimore, USA, Waverly Press. Chalmers, C. H. (1962) Bacteria in relation to the milk supply, 4th edn. London, Edward Arnold. Gasson,M. J. and Davies, F. L. (1984) The Genetics of Dairy Lactic Acid Bacteria, Chapter 4. In Advances in the microbiology and biochemistry of cheese and fermented milk. Amsterdam, Elsevier. Gotz, F., Sedewitz, B. and Elstner, F. (1980) Oxygen utilization by Lactobacillus plantarum Arch.
Microbial.
125,209-214.
International
Commission on Microbiological Specifications for Foods (ICMSF) (1980) Microbial ecology of foods vol. 2 factors affecting life and death of micro-organisms. New York, Academic Press. Lunder, T. L. (1979) Tea: History-Botany-Chemistry-Manufacture-Nutritional Value. Centre de Documentation Technique, Nestec. Mossel, A. A. and Tamminga, S. K. (1980) Methoden voor het mikrobiologisch Onderzock van Levensmiddelen. The Netherlands, Nordervliet, Zeist. Milieux et reactifs de laboratoire Pasteur (1981) Institut Pasteur, Department Production. France, Marnes-La-Coquette. Rehm, H. J. and Reed,G. (1983)Food and Feed Production with Micro-organisms, Chapter 5, pp, 578-586. Verlag Chemie, Weinheim, BRD. Rogosa,M. and Sharpe, M. E. (1959) An approach to the classification of lactobacilli. J. Appl. Bacterial
22, 329-340.
Vanos, V. and Bindschedler, 0. (1985) The Microbiology of instant coffee. Food Microbial, 2, 187-197. Vanos, V. and Cox, L. (1986) Rapide Routine Method for the Detection of Lactic Acid Bacteria among Competitive Flora. Food Microbial, 3, 223-234.
The microbiology
of instant tea
V. Vanes*, S. Hofstaetter
and L. Cox
Nest16 Products Technical Assistance Company, Central Quality Assurance Laboratory, Au. Nestle’, 55 CHl800 Vevey, Switzerland Received
15 October 1986
The microbial ecology of an instant tea line was studied using samples from different points of the line. Only lactic acid bacteria were found at certain manufacturing stages. The main contaminant was identified as Lactobacillus plantarum. As no other bacteria were found in this line, lactic acid bacteria may be considered as quality ‘indicators’ and their determination might be a means to monitor Good Manufacturing Practices (GMP). In addition, the total tea solids (TS) concentration that inhibits the growth of certain isolated L. plantarum strains was determined on the instant tea line. A TS concentration of 60% inhibited these organisms during48 h. The same TS concentration did not inhibit the growth of a non-identified cluster of Lactobacilli isolated from the same line. The bacteriostatic effects ofpure theophylline andpure theobromine on thegrowth of L. plantarum were also examined. No bacteriostatic effect in the presence of either compound was observed at normal concentration or at concentrations higher than those calculated to be normally present in tea lines. The standard aroma profile of an instant tea solution was modified as a result of inoculation with a wild strain of L. plantarum. This implies that heavy contamination and fermentation by such organisms might result in products with altered aroma profiles that are unacceptable to the consumer. It should be noted that altered tea aroma might eventually be used as an indicator of microbial build-up by analysing certain of the altered aroma components. Further work is nee&d to investigate this possibility. Challenge tests with known pathogens and spoilage organisms revealed that, although pathogens were unable to multiply in instant tea solutions, spoilage organisms could grow or survive and thus might pose problems.
Introduction Despite the wide consumption of instant tea, its microbiology is so far practically unknown. The objective of our study was to detect and identify the bacteria present at various points of a tea line and, according to the results thus obtained, to define quality ‘indicator’ * Present address: Nestle products Technical Assistance Company, Central Quality Assurance Laboratory, Av. Nestle, 55 CH1800 Vevey, Switzerland. (To whom correspondence should be addressed.) 0740-0020/87/010019
+ 15 $03.00/0
micro-organisms which might be used to monitor the hygiene aspects of Good Manufacturing Practices (GMP) in an instant tea factory. It is well known that tea contains elements such as alkaloids (the meth ylated derivatives of purine: caffeine, theobromine and theophylline) and catechins (flavonols), which may have a bacteriostatic effect (Lunder 1979). Caffeine levels vary between 3 and 5% in tea leaves and in manufactured tea (Lunder 1979) and are at about 4% in fresh tea shoot tips (Rehm and Reed 1983). 0 1987 Academic
Press Inc. (London)
Limited
20
V. Vanos et al.
Theophylline is present in the range of 0.23 to 0.44 mg per 100 g, and the theobromine content is approx. 50 mg per 100 gin manufactured tea. Flavonols (mostly catechins) constitute about 20% of the dry weight of tea leaves (Lunder 1979). Tea infusions are inhibitory to microorganisms, having a bacteriostatic effect in vitro on Salmonella typhi, Shigella paradysenteriae, Shigella dysenteriae, Staphylococcus aureus, Vibrio cholera and Leuconostoc mesenteroides (Lunder 1979). Little is known with regard to the effect of the concentration of natural inhibitors in tea on bacteria in general, especially those found in instant tea or potential spoilage and lines, pathogenic micro-organisms. For this reason, an attempt has been made to determine the tea constituents with the most inhibitory effects and to find out at what TS concentrations the tea solution completely inhibits growth of the predominant microflora. Among the alkaloids, the bacteriostatic effect of caffeine on certain bacteria was discussed in a previous article (Vanes and Bindschedler 1985). In the present study, the bacteriostatic effects of pure theophylline and theobromine have been examined as well as those of catechins as part of the examined tannin complex. The growth kinetics of some pathogenic and non-pathogenic bacteria in reconstituted soluble tea have also been studied. In this context potential
Table
1. Colonies
taken
from
different
hazards related to tea consumption have been considered. The modification of the standard aroma profile by controlled fermentation was also investigated.
Materials and Methods Microbiological profile of instant tea Ten samples from different points of a tea line were examined in duplicate (see Table 1, where only the points of the tea line which had presence of bacterial flora, are mentioned) using Chalmers’ medium (Vanos and Cox 1986) modified at our laboratory. Two well-dried plates per dilution were inoculated by surface spreading. One of the plates was covered with 8 ml of 15% water agar to obtain better ‘micro-aerophilic’ conditions, which are necessary for the detection of certain lactic acid bacteria. Plates were then incubated at 30°C for 72 h. In order to classify the isolated bacteria into groups, all colonies were first purified twice at 30°C on Plate Count Agar (PCA) OXOID CM 325 containing 1.5% sterile skim milk OXOID L31 (10% reconstituted). Afterwards, the following tests were carried out: 1. Cat&se test: Reaction with 30% H202 to detect the presence of the enzyme catalase. 2. Gram staining: Observation of the cell morphology and the possible presence of spores. 3. Growth tests in MRS broth at diferent temperatures: 10,30,37 and 45°C during 48 h of incubation. 4. Homo-heterofermentation test: To detect strains producing gasfrom glucosefermentation (Mossel and Tamminga 1980). 5. A.DJI. test: To detect the presenceof the arginine dihydrolase enzyme in the isolated strains (Institut Pasteur 1981). 6. Ability to ferment ribose(FLU&I 83860).
points
in the tea-line.
No. of sample Point of the tea-line 1
2 3 ii
Concentrated liquor after evaporation Distillate Clarified liquor in surge tank Concentrated liquor in surgetank Spray-mix in HP pump surgetank
Coloniestaken 66 5: 20 67
Microbiology To identify the Lactobacillus species, the following substrates were used: From MERCK: D-Arabinose (1494); L-Arabinose (1492); Dulcitol(5990); meso-Erythritol (3160); Esculin (842); D(+) Glucose (8342); meso-Inositol(4728); Lactose (7657); Maltose (5910); D-Mannitol (5982); D(+) Mannose (5984); Raffinose (7549); Salicin (7665); L(+) Rhamnose(4736); D-Xylose (8692). From FLUKA: D( +) Galactose (48260); Gentiobiose (Amygdalose) (48790); Fructose (47740); D(+) Melibiose (63630); D(+) Saccharose(84100); DSorbitol(5530). The ability of the isolated strains to ferment these substrates was tested in MRS broth in which glucosewas replaced by the respective substrate at a concentration of 2%, with 0.001% bromocresolpurple as pH indicator. Becausecertain sugar fermentations are in general plasmid-linked (Gasson and Davies 1984), a positive identification was recorded if 20 out of the 21 substrates gave typical results, offering a confidence limit of 95%. Furthermore, the configuration of the lactic acid was examined by optical rotation, asa complementary test to identify certain strains. The API 20 STREP identification kit was usedto identify the Streptococcus strains. Strains used Lactobacillusplantarum Lactobacillus sp.
(cluster F) Streptococcus fwcium Pediococcus pentosaceus Bacillus cer&s Escherichia coli Salmonella typhimurium Staphylococcus aureus Streptococcus faecalk Saccharomyces cerevisiae
Culture
of instant tea
21
Media For the purification of the non-lactic strains, the following media were used: B.cereus: S. aureus: S. faecalis: E. coli: Salmonella
typhimurium: Saccharomyces cerevisiae:
M.Y. agar (MERCK 5267) + supplements (egg yolk + polymyxin) Baird Parker (OXOID CM 275) + supplements (eggyolk + tellurite) KF agar (DIFCO 0496-01-o V.R.B.L. agar (DIFCO 0012-01-05) B.G.A. (OXOID CM 329) O.G.Y.E. agar + supplement(OXOID CM 545)
For plate count determinations, the following media and incubation conditions were applied. Lactic strains: MRS agar, (OXOID CM 361); Incubation at 30°C for 72 h. Non-lactic strains: Plate Count Agar (PCA, OXOID CM 325) + 1% triphenyl tetrazolium chloride (‘ITC) from a 1% solution (DIFCO 3112-67-9) was used for all strains except Origins
QA laboratory, isolated from tea line QA laboratory, isolated from tea line QA laboratory, isolated from tea line QA laboratorv. isolated from tea line ” Lausanne2868 ATCC 25922 ATCC 14028 ATCC 25923 ATCC 206 Linor (NESTEC)
conditions
The non-lactic strains were first purified on selective media before being cultured in Trypticase Soy Broth (TSB BBL 11043) at 37°C. Lactic strains were cultured in MRS broth at 30°C. To obtain cultures with maximum activity, each strain was subcultured three times in the corresponding broth before use. For the experiments, all strains were taken at the late log phase.
culture collections
B. cereus and S. aureus where the respective
selective media were used. Incubation: 37°C for 24 h. Dilutions were made in tryptone-salt (l-8.5 g-1). Inoculation techniques for solid media Double layer (overlay the poured inoculated layer with the same medium) for MRS medium; poured plates for the PCA medium and surface spreading for M.Y. and BairdParker media.
22
6. Vanos et al.
Preparation of soluble tea For the preparation of a soluble tea solution, 2 g of instant tea powder were added to 100 ml of distilled water at 100°C; after 10 mins the tea was cooled to 37°C. This method resulted in less than 10 colony forming units (cfu) per ml.
sterile 2% TS tea solutions. In 10 and 12% solutions the pH was 7.7. The growth of L. plantarum was examined in these solutions. The same assay was repeated by adjusting the pH to 6 in the 10 and 12% tannin solutions. Growth of pathogenic and spoilage micro-organisms The solutions of instant tea (2%) were inoculated with the different micro-organismsat a level of 104-107 cfu ml-i. They were incubated at 30°C in all solutions during 48 h. At intervals of 4, 8, 16, 24 and 48 h they were plated in the corresponding selective media.
Bacteriostatic effect of theophylline Despite its low concentration, theophylline could contribute synergistically to the bacteriostatic effect of instant tea. Sterile pure theophylline (FLUKA 88308) was added to a final concentration of 0.3 mg ml-l to 2% TS instant tea solution. This theophylline concentration is about 200 times higher than in a normal instant tea Preparation and fermentation of soluble tea solution with a TS content of 60%. The solution was inoculated with L. plan- The tea powder was reconstituted at 2% in tarum (lOa--lOa cfu ml-i) and incubated at sterile distilled, boiling water in four screw30°C during 48 h. At intervals of 4,8, 16, 24 cappedbottles and then cooledto 37°C. Two of and 48 h, L. plantarum colonieswere counted them were inoculated with the same inocuin the corresponding selective medium. lum from the L. plantarum strain isolated from the tea line to produce 10-10s cfu ml-i. These bottles, with one control bottle, were Bacteriostatic effect of theobromine incubated at 30°C for 48 h. The other control Despite its low concentration, theobromine bottle was analysed immediately for the could also contribute synergistically to the aroma profile. bacteriostatic effect of instant tea. Since the pure theobromine used (FLUKA Screening of the aroma profile of 88304) was not water-soluble, a solution of fermented soluble tea 5% was made in dimethylsulfoxide (DMSO) The analyse the aromas of fermented tea, a (MERCK 802912). DMSO did not inhibit gas chromatograph HP5880 was used with a lactic acid bacteria. This solution was then headscapetrapping apparatus (J. Rectorit), added to 2% TS instant tea with a final (trap 6 min, N2 = 60 ml min-1, GC: 15 min concentration of 0.3 mg ml-i, the normal 180°C(2°C min-i), P = 1 bar, split = 10 ml concentration of 60% TS instant tea. The min-1). The analysis was performed in duplisolution was inoculated with the samestrain cate for fermented and non-fermented soluble of L. plantarum and the above procedure was tea. repeated. Water activity (ad measurement Bacteriostatic effect of catechins The a, was measured with a NOVASINA The tannins found in the tea leaves are not a EEJA-3 apparatus. Calibration for the 0.902 single chemical compound but rather a mix- value was made at 25°C by using saturated ture of polyphenols representing as much as BaC12. 30% of the dry weight of the leaves (Lunder 1979). pH measurements In this mixture of polyphenols, flavonols (known as catechins) represent about 83% The pH was measured with a digital pH(Lunder 1979). Since the catechin complex Meter (E 532 Metrohm Herisau). was not commercially available, its bacteriostatic effect on Lactobacillus growth was examined indirectly by using a 12% tannin Results solution which was obtained from the factory as the maximum concentration of tannins A total of 212 colonies were picked at found in the tea line. Dilutions of this random (picking the square root of the solution were made at 2, 4, 5, 8, and 10% in number of the colonies of the examined
V = variable.
-ve
-ve
Pediococcus
Atypieals
Streptococcus
-ve
Streptobacteria (Rogosa’s classification)
Lactobacillaceae
Streptococcaceae
-ve
Group
Catalase
of the groups
Family
1
Table 2. Characteristics
+ve non-spore forming rods +ve cocci +ve cocci in tetrads
+ve non-spore forming rods
Gram staining
of bacteria
-
+
+
f
10
+
+
+
f
+
4
+
+
37
-
+
V 50% of the strains were +ve -
45
from the tea-line Growth at (“C)
30
isolated
-ve
-ve
--Ye
-ve
Gas production from glucose
+ve
+ve
-ve
-ve
of arginine dihydrolase
A.D.H. presence
as well as the number
-ve
-ve
-ve
+ve
fermentation
Ribose
of isolated
iii
4
9
30
169
isolated strains
Number of
strains.
24
V. Vanos et al.
plates)
from the plates for identification.
Table 3 shows the substrate utilization profiles of the identified and unidentified strains. Table 4 summarizes the different points of the tea line, from which the identified species were isolated. The results of growth studies at different TS concentrations are presented in Figures 1, 2, 3, 4 for each species of isolated bacteria (L. plantarum, Lacto-
Apart from some samples which did not carry any bacteria, only lactic acid bacteria were detected in the positive samples. Table 1 summarizes the numbers of bacteria isolated from the various samples as well as the number of colonies taken for identification. Table 2 lists some characteristics of the groups of bacteria isolated from the tea line, as well as the number of strains isolated per group.
Table 3. Use of substrates
by the strains
Clusters of Lactobacillus Substrates
bacilli-cluster saceus) .
F, S. faecium,
The growth of the dominant
isolated
Cluster of Streptococcus strains (number of strains)
Cluster of Pediococcus strains (number of strains) A (41
A(1201
B(30)
c(10)
D(1)
E (1)
F (281
A (8)
Amygdalose Arabinose D Arabinose L Dulcitol Erythritol Esculine Fructose Galactose Glucose Inositol Lactose Maltose Mannitol Mannose Melibiose Raffinose Rhamnose Salicine Sorbitol Sucrose Xylose D
+
+
+
+ + + + + + + + + + + + + + -
+ + + + + + + + + + + + + + + + -
+ + + + + + + + + + + + + + + + -
+ -
+ + + + + + + + + + + + + + +
+ + + + + + + + + + + + + + + -
+ +
Lactic acid configuration
DL
Identified species
L.plantlVU?Tl
Atypical but very closeto L. plantarum
organism,
from the tea-line.
strains (number of strains)
Not examined
P. pento-
+ + + +
-
+ + + + + + + + + +
Not Not Not examined examined examined Atypicals
S. faecium
P. pentosaceus
Microbiology Table 4. Percentage
of species isolated
from different
of instant
Isolated species (o/oof total isolated bacteria)
Concentrated
L. plantarum (84) Atypical lactobacilli (16) P. pentosacew (80) S. faecium (20) L.plantarum (40) Atypical lactobacilli (20) S. faecium (40) L. plantarum (61) Atypical lactobacilli (39)
Distillate Clarified liquor in surge tank Spray mix in HP pump surge tank
L. planturum, is shown in Fig. 1. Inhibition began at 10% TS and was complete at 60% Ts. The growth of the Lactobacilli-cluster F is shown in Fig. 2. Inhibition was not complete, even at 60% TS, but growth was quite slow at this. These bacteria grew better at 10, 20 and 30% TS than at 2% TS. The growth of S. fuecium was completely inhibited at 30% TS (Fig. 3). This strain grew best at 10% TS, whereas at 2% TS a rapid die-off occurred after 16 h incubation. The growth of P. pentosuceus was inhibited even at 2% TS (Fig. 4). This strain was a mesophile and did not grow at 45°C after 48 h incubation.
The growth dynamics of the dominant species L. plantarum in the presence of pure theophylline and theobromine are presented in Figs 5 and 6. No inhibition occurred at 0.3 mg ml-l during 48 h with either compound. The bacteriostatic effects of different tannin concentrations on the growth of the dominant strain L. phturum are shown in Figs 7 and 8. At 10 and 12% of tannins, growth was inhibited during 24 h at pH 7.7 but not at pH 6. The growth kinetics of E. coli and S. typhimurium are presented in Figs 9 and 10. After incubation at 37°C for 48 h, E. coli could not be detected. Salmonellae
TS % * --__-,o . . . . . . . . . zoso------
I
60----I 4
I 8
I 16
I 24
I 48 Tome
Fig. 1. Growth
of L. plantarum
25
points of the tea-line.
Point of the tea-line liquor after evaporation
tea
(h)
in instant tea at different TS concentrations.
-
-
26
V. Vanos et al. TS %
I
j
I I
I
I
4
8
I 6,
48
24 Ttme
Fig. 2. Growth
60-
I
I
of cluster F of lactobacilli
(hi
in instant tea at different TS concentrations.
TS %
8 7
:
t
.,..........
. .._...................
...
2
. . .. . . .. ..
---__
I
Tome (h)
+ Fig. 3. Growth
of S. foe&m
in instant tea at different TS concentrations.
TS % 8-
73 ‘;
6-
/ /
5-
1
2
-----
IO ’ 20 30 40 50
1
1 4
I 8
I I6
1 24
I 48 7me
Fig. 4. Growth
r--1
ih i
of P. pentosaceus in instant tea at different TS concentrations.
Microbiology
Fig. 5. Growth of L. plantarum in instant tea at 2% TS (- - -) and in instant tea at 2% TS containing O-3 mg ml-1 of theophylline G--h
‘r
4
8
16
24 Time
of instant
tea
27
decreased rapidly after 24 h and were not detected after 48 h. Under the same conditions, S. aureus was not detected either (Fig. 111, whereas the number of S. faecalis remained constant during 48 h (Fig. 12). B. cereus colonies were not detected even after 16 h (Fig. 131, whereas the number of S. cereuisiae remained constant during 48 h of incubation (Fig. 14). The instant tea solution, inoculated with L. planturum and incubated at 30°C for 48 h, clearly showed qualitative changes in the aroma profile (Figs 15, 16). Unidentified new peaks with retention times (R,) of 30 and 68 min were detected in duplicate samples, and the peak with Rt 20 min had decreased. The sterile instant tea solution, incubated at 30°C for 48 h, did not show any modification of the standard aroma profile in comparison with the non-incubated sterile instant tea solution (Figs 17, 18).
48 ( hi
Fig. 6. Growth of L. pluntarum in instant tea at 2% (----) and in instant tea at 2% containing O-3 mg ml-l of theobromine G--1.
Discussion In the examined samples taken from different points of the tea line, the predominant microflora was found to be entirely constituted by lactic acid bac-
*.----
4-
-
-
6---
8--IO-
Fig. 7. Growth of L. plantarum in instant tea (2% TS) at different concentrations 10 and 12%, the pH was found to be 7.7.
-
-
of tannins. At
28
V. Vanos et al.
Fig. 8. Growth of L. plantarum
in instant tea (2% TS) at different concentrations of tannins. The pH was adjusted to 6 at 10 and 12%.
) sterile distilled water, (- - - -1 instant tea
Fig. 9. Survival ofE. coli strain ATCC 25922. (2%.
$-:7:-i ,,,_,,_,,___,: 4
l
8
16
24 Time
Fig. 10. Survival of Salmon&z water, (- - - -) instant tea 2%.
typhimurium
teria, mainly of the genus Lactobacillus. L. plunturum was the prevalent identified species, probably because of its ability to tolerate a relatively high concentration of oxygen, compared to the other species (Gotz et al. 1984). The same
48
(h)
strain ATCC 14028. (-)
sterile distilled
species had also been predominant in an instant coffee line as observed in an earlier study (Vanos and Bindschedler 1985). Although the conditions for growth ofLactobacilli (temperature, acid pH, presence of vitamins, amino acids
Microbiology
y.
4
I6
8
l ‘.
of S. aureus
8
16
16
‘1
‘.
b48
8
I 48 (hi
sterile distilled water, (- - - --I instant
24 Time
(h)
Fig. 13. Survival of B. cereus strain Lausanne 2868. (-) instant tea 2%.
sterile distilled water, (- - - -)
7 l ; z
61 5r). 4<
,*
-0-e - -.----
..-+--
L----L-, 4
8
16
cerevisiue
____-_--
.---
24 Time
Fig. 14. Survival of Saccharomyces (- - - -1 instant tea 2%.
29
sterile distilled water, k----I
24
Fig. 12. Survival of S. fuecalis strain ATCC 206. (-) tea 2%.
8
‘1
_----------_
Time
4
‘\
tea
ih)
strain ATCC 25923. (-)
S----S
4
‘,
24 Time
Fig. 11. Survival instant tea 2%.
‘,
of instant
-0
1
48 (h)
strain ex Linor. (-
) sterile distilled water,
30
V. Vanos et al. NESTEA
CONTROL
SAMPLE
(WITHOUT
j
INCUBATION)
12.70
8.71
Fig. 15. Aroma profile of a sterile instant tea solution.
I
NESTEA
+ L PLANTARUM
IO*00
20~00
30.00
Top Raw: BTHEOZ Bottom Row: BTHE03
(O-O-80.0) (O-O-80.0)
(Enlarged (Enlarged
CPOO Relative
40.00
(INCUBATION
50.01
48
H)
60.01
70.01
Plots x 30.0) x 30.0)
Fig. 16. Aroma profile of an instant tea solution inoculated with L. plantarum.
NESTEA
CONTROL
SAMPLE
(WLTHOUT
INCUBATION)
3.06
8.76 7.69
Fig. 17. Aroma profile of a sterile instant tea solution.
80.1
Microbiology INCUBATED
12.70
- _
I I.70
-
IO-70
-
9.70
-
8.71 7.71
i 0.00
NESTEA
SAMPLE
(INCUBATION
48
of instant tea
31
H)
1Ii IO.00
20.00
Relative Plots Top Row: BTHEOI Bottom Row: BTHE02
(0.0-80-O) 10*0-80.0)
30.00
( Enlarged i Enlarged
40.00
50.01
60.01
70.01
SO4
x 30.0) x 30.0)
Fig. 18. Aroma profile of the same sterile instant tea solution incubated at 30°C for 48h. and sugars) are less restrictive in a tea line than in a coffee line, the diversity of the detected Lactobacilli species was not the same. Of the 212 isolated strains, 93% were identified as Lactobacilli, 4.5% as Streptococci and 2.5% as Pediococci (Table 2). In the Lactobacilli group, 60% were identified as L. plantarum and the rest as atypical (Table 3, cluster A). The atypical Lactobacilli were classified into clusters (Table 3, clusters B, C, D, E, and F) according to their ‘substrate use’ profiles. Clusters B, C, D, and E were very close to the L. plantarum substrate profile, differing in the use of only two or three substrates. Cluster F (28 of the strains) differed considerably from the L. plantarum substrate profile. These strains did not ferment arabinose L, mannitol, melibiose, raffinose, sorbitol or sucrose, but only rhamnose. They were very similar to the species L. casei, Subsp. rhumnosus, but with a confidence limit below 90%. All the isolated Lactobacilli were mesophiles, of which 85% were classified as Streptobacteria as indicated in Table 2 (Rogosa and Sharpe 1959). All Lactobacilli grew at 10°C during 48 h incubation, and 50% of the Streptobacteria strains grew at 45°C. Therefore, to prevent multiplication of
Lactobacilli in the tea line, temperatures should be either higher than about 50°C or lower than 10°C. The absence of heterofermentative Lactobacillus species among the isolated strains might reduce the possibility of gas production as a problem in such a line, if abundant multiplication occurs during the weekend in insufficiently cleaned pipes. The presence of group D Streptococci, although they may survive in tea solutions, does not seem to represent a health hazard, due to their low incidence (4.5% of all isolated strains). The species present in this group was S. faecium. The examined strains could grow at 45°C. Other strains, however, can grow at 52°C. S. faecium was detected only in two samples from the distillate and from the concentrated liquor in the surge tank. As this strain had a very low incidence (4.5%) among the identified flora, it cannot be considered as a quality indicator of the tea line. Probably it does not originate from the line, but could be due to airborne post-contamination at the moment of sampling. There was a very low incidence of pediocoeci (only 2.5% of the total isolated flora) and therefore their presence would not seem to pose any problems. The growth of L. plantarum was com-
32
V. Vanos et al.
pletely inhibited in a tea solution of 60% TS. In experiments with 40-45% TS, inhibition of the growth persisted only for 16 h. This could mean that the growth of L. phtarum will not be completely inhibited under normal processing conditions in a tea line, where the maximum amount of TS in some parts of the line is 4M5%. Lactobacilli-cluster F tolerated the natural bacteriostatic elements of the tea, even at a high concentration. This group of Lactobacilli seems to represent a more important spoilage hazard for the tea line than L. plantarum. A P. pentosaceus strain was isolated from only one sample of the tea line and died off rapidly in instant tea solutions. It is possible that this species was not a true contaminant of the examined tea line, and that its presence in that particular sample resulted from accidental contamination during sampling. As mentioned before, L. plantarum was completely inhibited at 60% TS, at which concentration water activity was O-96 and pH 5-3. Neither of these values inhibits the growth of Lactobacilli (ICMSF, 1980). Therefore, the inhibition at 60% TS might have been caused by the high concentration of catechins and the alkloid complex ‘caffeine + theobromine + theophylline’. A bacteriostatic effect of caffeine on the growth of L. plan&rum has already been well demonstrated in a previous study (Vanos and Bindschedler 1985). In the present study, the alkaloids theobromine and theophylline have been investigated for their possible bacteriostatic effects on the dominant strain, L. plantarum. No bacteriostatic effect was observed with either compound, not even when theophylline was used at a concentration 200 times higher than normal. Therefore, the complex Ylavonols (cate-
chins) + caffeine’ seems to be the only natural inhibitor of Lactobacilli in instant tea. Complete inhibition of L. plantarum growth was obtained with a 12% tannin solution. The pH of that solution was 7.7 (Fig. 7). When pH was adjusted to 6.0, inhibition persisted only for 16 h (Fig. 8). Thus, the bacteriostatic effect of the tannin complex might be pH-dependent. However, at pH values higher than 7 Lactobacilli are inhibited (ICMSF 1980). A synergism between pH and tannin bacteriostasis is therefore difficult to confirm. The growth kinetics of pathogenic bacteria (Figs 9, 10, 11, 13) in instant tea solutions show that tea liquor is not a suitable medium for active growth of these strains, even if larger inocula are used than might occur naturally in a tea line. The Streptococci group D (S. faecium and S. fuecalis) and the yeast S. cereuisiae could represent a spoilage hazard for the tea line, since they are able to grow in this substrate. Lactic acid bacteria can be considered as ‘indicators’ of the quality in a tea-line, because no other bacteria such as coliforms, spore formers, etc were detected. However, the extent of their presence and its true significance remain to be investigated. The inability of certain pathogenic organisms to grow in instant tea solution indicates that public health hazards are most unlikely to arise in relation to this product. Moreover, further investigations into the modification of tea aroma by Lactobacilli are required before any final conclusion may be drawn regarding the significance of these micro-organisms for the sensory quality of instant tea.
Microbiology of instant tea 33 Acknowledgements We wish to thank Dr J. C. Spadone, Laboratory of Organic Chemistry, Central Research Department of NESTLE (Vets-ehez-les Blanc, Switzerland), for providing the aroma profiles of the fermented and non-fermented instant tea solutions.
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Commission on Microbiological Specifications for Foods (ICMSF) (1980) Microbial ecology of foods vol. 2 factors affecting life and death of micro-organisms. New York, Academic Press. Lunder, T. L. (1979) Tea: History-Botany-Chemistry-Manufacture-Nutritional Value. Centre de Documentation Technique, Nestec. Mossel, A. A. and Tamminga, S. K. (1980) Methoden voor het mikrobiologisch Onderzock van Levensmiddelen. The Netherlands, Nordervliet, Zeist. Milieux et reactifs de laboratoire Pasteur (1981) Institut Pasteur, Department Production. France, Marnes-La-Coquette. Rehm, H. J. and Reed,G. (1983)Food and Feed Production with Micro-organisms, Chapter 5, pp, 578-586. Verlag Chemie, Weinheim, BRD. Rogosa,M. and Sharpe, M. E. (1959) An approach to the classification of lactobacilli. J. Appl. Bacterial
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Vanos, V. and Bindschedler, 0. (1985) The Microbiology of instant coffee. Food Microbial, 2, 187-197. Vanos, V. and Cox, L. (1986) Rapide Routine Method for the Detection of Lactic Acid Bacteria among Competitive Flora. Food Microbial, 3, 223-234.