Abstract
In Sri Lanka, several tea grades are produced in factories located in different agro-climatic regions within three geographical elevations. The study aimed to determine the microbial quality of different tea grades and composite tea samples obtained from factories situated at diverse locations. The average APC, yeast & mould counts and coliforms in different tea grades ranged from 3.4 × 103 to 2.0 × 104 cfu/g, 4.8 × 102 to 2.5 × 103 cfu/g and 0.005 to 3.9 × 101 Most Probable Number (MPN)/g respectively. The tea samples collected from different factories had mean values of APC and yeast & mould as 5.3 × 103±1.3 × 103 cfu/g and 9.7 × 102±1.9 × 102 cfu/g. Escherichia coli (E.coli) and Salmonella were not detected either in tea grades or in composite samples. The identified microorganisms in tea samples belong to phyla Firmicutes, Proteobacteria, Ascomycota, Basidiomycota and Zygomycota.
The samples collected from the mid country elevation had the highest counts of APC and yeast & mould counts were high in the low country elevation. More than 70 % of the tested samples comply with the SLTB guidelines given for the microbiological quality of black tea. The distribution of bacterial, yeast and mould and coliform densities of tea were significantly variable with respect to geographic areas.
Keywords: Black tea, Tea grades, Microbial quality, Agro climatic regions, Elevations
1. Introduction
Tea is one of the oldest beverages in the world and this non-alcoholic, caffeine-containing plant infusion is popular due to its taste, aroma and valuable physiological properties. The quality of tea primarily depends on the chemical composition of the harvested tea leaves as well as the way tea leaves are handled, processed and stored [1]. Additionally, many other factors such as cultivar species, season, age of the tea leaves, climate and horticultural practices (including soil, water, minerals, fertilizer and geographical location) may influence the chemical composition of the tea leaves [[2], [3], [4]].
In general, teas are divided into four categories based on the tea manufacturing process: Green tea (unfermented), Oolong tea (partially fermented), Black tea (fermented) and post-fermented dark tea [5a], [5]. In Sri Lanka, tea-growing regions are divided based on geographical elevation into high grown (upcountry), medium grown (mid-country) and low grown (low country) areas and these regions experience distinct temperature variations. These three elevations have been further sub-divided into seven agro-climatic regions. There is abounding evidence in literature to indicating that the flavours and aroma of teas from each elevation are influenced by the climatic conditions [6,7]. The climatic variation in each tea-growing district has led to the production of different tea grades with some being unique to each agro-climatic region.
After plucking the leaves, the processing steps that followed are withering, rolling, fermentation and firing. The final product is sorted, graded, packed, and stored [8,9]. Microbial contamination in tea can occur during various stages, including from the phylloplane flora and then during the plucking, withering, rolling and fermentation processes. However, the contamination is reduced during the drying process. However, poor post-handling steps such as grading, packing and storing can lead to additional microbial contamination of the graded tea [9]. Tea is produced as a dry product with low moisture content, which restricts the growth of bacteria. Using hot water for preparing tea brew may reduce the risk of bacterial contamination, although bacterial spores may still be present [10].
The dry tea leaves are vulnerable to microbial contamination and the presence of various bacteria and moulds poses a risk to the consumer's well-being [11]. In recent years, attention has been focused on the microbiological contamination levels of black and green teas [[11], [12], [13], [14]].
Researchers have shown that the quality of black tea is influenced by various factors, including the region of production and climate [17a], [17b] as well as the genetics of the tea plant [15]. In Sri Lanka, the climatic conditions in tea-growing regions vary from one another and these climatic variations are reflected in the diversity of quality characteristics in tea. The results reported by Jayasekera et al. (2011) [2] indicated significant variations in the antioxidant activity of tea samples collected from different tea-producing regions in Sri Lanka.
The revelation of the effects of different climate conditions on tea production has led to the manufacturing of a variety of tea products. Some of these products are unique to each region and the pricing is also influenced by these factors. However, there is limited information available regarding the microbiological quality and microbiota of tea produced in different agro-climatic regions in Sri Lanka.
Climatic variations can affect both the microbial loads and the specific microorganisms present in tea products and this aspect has not been thoroughly investigated. Therefore, the present study was conducted to assess the microbiological quality parameters in black tea and explore their variations based on agro-climatic regions and elevations. Additionally, this study examined the presence of different microbial loads in selected graded black tea samples. It is expected that the results will be valuable for manufacturers in taking necessary actions to prevent contamination. The data is also intended to support the development of suitable strategies to comply with the guidelines set forth by the Sri Lanka Tea Board.
2. Materials and methods
2.1. Selection of tea factories
The selection of tea factories was carried out following an information survey that considered their location, the availability of quality certificates and the different types of tea grades produced by 754 tea factories. Data was obtained from Registered Tea brokers and the Sri Lanka Tea Board (SLTB). The tea grade nomenclature is provided by the Tea Exporters Association in Sri Lanka (2017) [16]. Data collected during the years 2015–2017 was used in the information survey.
Out of 722 operational tea manufacturing factories, 28 were selected for the study based on their agro-climatic regions and the availability of quality certificates (ISO 22000, HACCP or other accepted quality certificates). These selected factories were further divided into groups: 14 from low country elevation, 10 from up country elevation and 4 from mid-country elevation (Table 1). The study also documented various tea grades produced in these factories.
Table 1.
Selected tea factories, their code numbers, agro-climatic regions, elevations and number of samples collected.
| Elevation | Agro-climatic region | Factory codes and number of samples collected | ||||
|---|---|---|---|---|---|---|
| Low country | Ruhunu | NLR04 (30) | NLR05 (30) | NLR07 (30) | NLR12 (30) | NLR14 (30) |
| YLR02 (30) | YLR03 (30) | YLR06 (27) | YLR13 (30) | YLR15 (30) | ||
| Sabaragamuwa | NLS08 (27) | NLS28 (30) | YLS01(30) | YLS09 (30) | ||
| Mid country | Kandy | NMK23 (27) | NMK25 (24) | YMK22 (12) | YMK24 (30) | |
| Up country | Dimbula | YUD16 (12) | YUD17 (15) | YUD21 (15) | ||
| Nuwaraeliya | YUN10 (18) | YUN11(27) | YUN20 (27) | |||
| Udapussallawa | YUP18 (24) | YUP19 (15) | ||||
| Uva | YUU26 (27) | YUU27 (27) | ||||
[code no = 1st digit -availability of quality certificates: Y= Yes or N No; 2nd digit – Elevation; 3rd digit – Agro-climatic region; 4th digit – factory number; example NLR 04 (N – No certification, L –Low country, R – Ruhuna Agro-climatic region; 04 – given factory number).
The number of tea grades produced in factories located in each agro-climatic region was calculated as follows;
| % of factories produced BOP* = |
| Number of factories produced BOP in the up-country elevation ×100 |
| Total number of factories located in the up-country elevation |
(* = a tea grade named Broken Orange Pekoe).
2.2. Collection of different tea grades
Out of the 28 factories, ten different tea grades were selected for this study and coded as follows: GA, GB, GC, GD, GE, GF, GG, GH, GI and GJ each code corresponding to one of the ten tea grades. All the selected tea grades were not produced by all the selected factories. A few selected tea grades are given in Fig. 1.
Fig. 1.
Shapes of different Tea Grades.
Each factory was visited three times at different time intervals to collect samples during the three-year research period. In one visit, 238 samples of investigated tea grades were collected from 28 factories [i.e. GA (28), GB (21), GC (26), GD (22), GE (22), GF (15), GG (28), GH (25), GI (27) and GJ (24)]. During this period, seven hundred and fourteen tea samples (238 × 3) were collected and tested to represent the seven tea-growing agro-climatic regions in three different elevations of Sri Lanka.
Approximately 200 g of graded tea samples were collected into sterile bags separately. All the samples were collected within 24 h of the manufacturing process. Microbiological analysis of the samples were performed in three replicates.
2.3. Sample preparation and microbiological analysis
In order to estimate the population of the microorganisms in graded tea samples, 10.0 ± 1.0 g of sample was measured into a sterile bag (400 mL) and mixed with the diluent (90 mL of peptone salt water). The prepared liquid mixture was shaken for 60 s in a stomacher (INTERSCIENCE, BAG MIXER 400) and the suspension was used for the preparation of further dilution series [17] for enumeration of each group of microorganisms.
The enumeration of yeasts and moulds was performed as given in ISO 21527–2: 2008 [18] standard procedure. Surface inoculated plates were prepared by using Dichloran 18 % Glycerol agar (DG 18) (OXOID, UK). After the aerobic incubation of plates at 25 ± 1 °C for 7 days, the number of yeasts and moulds colonies was counted and calculated as given in the standard. The results were reported as Colony-forming Units (CFU) per gram of tea sample.
The initial suspension and decimal dilutions (1 mL) prepared from each tea sample were dispensed into sterile Petri dishes in duplicate and mixed with Plate Count Agar medium (OXOID, UK). After the aerobic incubation of plates at 30 ± 1 °C for 72 ± 3 h, the number of bacteria per gram of tea sample was calculated and results were reported as colony forming units (cfu) of Aerobic Plate Count (APC) per gram of tea [19].
According to the procedure given in ISO 4831:2006 [20], coliforms were detected by Most Probable Number (MPN) method. The method followed was the multiple tube technique. Initial suspension (10 mL to double strength medium contained tubes, 1 mL to three single strength tubes and 0.1 mL to another three single strength tubes) was transferred to lauryl sulfate tryptose broth (OXOID, UK) and incubated at 37 ± 1 °C for 48 ± 2 h. The presence of coliforms was confirmed by inoculation of brilliant green bile lactose broth with bacterial suspension which is incubated in the same condition. E. coli was confirmed by subculturing the positive lauryl sulfate tryptose broth tubes in EC medium incubated at 44 °C for 48 ± 2 h following indole free peptone water [21].
Salmonella was detected according to ISO 6579–1:2017 [22]. Four successive stages were followed for the detection of Salmonella. Buffered peptone water (OXOID, UK). was used as pre-enrichment in a non-selective liquid medium. After the incubation at 37 ± 1 °C for 18 ± 2 h inoculums from pre-enrichment were transferred into two selective liquid media (Rappaport-Vassiliadis Soya Peptone Broth and Muller-Kauffmann Tetrathionate-Novobiocin Broth) (OXOID, UK) separately for enrichment. This was then followed by isolation of colonies on differential media (Xylose Lysine Deoxycholate agar and Brilliant Green Agar) (OXOID, UK). The selective colonies of presumptive Salmonella were subcultured and confirmed using biochemical tests (Triple Sugar Iron Agar, Urea agar, Tryptone broth and Lysine broth) and confirmed by serological tests (Salmonella ‘O’ antisera and ‘H’ antisera).
2.4. Identification of isolated microorganisms
The bacteria and fungi isolated from the different black tea samples were subjected to identification program which includes morphological, biochemical and molecular biological techniques. The bacterial colonies with different morphologies (circular/irregular form colonies, colonies with raised/flat/convex elevations, colonies with entire/undulate/curled margins) were subjected to specific biochemical tests as per Bergey's manual [23]. The isolated bacteria were stored in Plate Count Agar. Selected colonies were submitted for molecular biological analysis.
Morphologically different fungal colonies were stored on Rose Bengal agar and then the selected colonies were subjected to molecular analysis.
2.5. Extraction of DNA
A bacterial colony was suspended in 60 μl of sterile ultrapure water contained in 1.5 mL eppendorf tube. The mixture was kept in a preheated water bath at 95 °C for 5 min and centrifuged (microcentrifuge, MPW-150R) at 4000 rpm for 5 min. An aliquot (1 μl) of the supernatant was used as a template for PCR.
A rapid extraction method was used for the extraction of genomic DNA from yeasts and moulds colonies using the ZymoBIOMICS ™ DNA Microprep Kit (ZyMO RESEARCH, Cambridge bioscience, UK).
2.6. PCR amplification and sequencing
The isolated genomic DNA was amplified using primers obtained from IDT Technologies. Following primers which amplify the 16S bacterial rRNA were used for amplification of bacteria [5b], [24], [25].
27 F (5′- AGA GTT TGA TCM TGG CTC AG -3′) and.
1492 R (5’ - TAC GGY TAC CTT GTT ACG ACT T - 3’)
Amplification of ITS region of yeasts and moulds was performed using following primer pair [26,27].
ITS1F (5’ - TCC GTA GGT GAA CCT GCG G - 3’) and.
ITS 2R (5’ - GCT GCG TTC TTC ATC GAT GC - 3’)
The reaction mixture for amplification was a volume of 25 μL containing 1 x Taq buffer containing 1.5 mM MgCl2, 0.2 mM dNTP, 0.25 μM of forward and reverse primers, 0.5 U Taq DNA polymerase (abm, Canada) and 10–20 ng of genomic DNA using Quant Studio 3 (Applied biosystems by Thermo Fisher Scientific). Thermal cycling consisted of initial denaturation at 94 °C for 5 min, followed by 35 cycles of denaturation at 94 °C for 30 s, annealing at 52 °C (ITS) and 49 °C (16S) for 30 s, elongation at 72 °C for 60 s, and finally elongation at 72 °C for 7 min. In parallel, negative control was run without template DNA and visible PCR products was not detected.
The amplicons were resolved in a 0.8 % agarose gel containing 0.5 μgml−1 ethidium bromide (Multisub electrophoresis systems, Cleaver scientific Ltd, powerpro 300V). The separated bands were purified and custom sequenced at Macrogen Ltd, Korea. The sequences were analysed using Bio edit version 5.2 software. A blast analysis of the sequence against the redundant database at NCBI was carried out to identify the organism [28,29].
2.7. Statistical analysis
A three-stage nested design was applied for the analysis. Sample data was obtained from 28 factories representing seven agro-climatic regions and three elevations with six replicates. (Three samples were collected from each factory on 3 different days and each sample was duplicated). When the F ratio supports the rejection of the null hypothesis, indicating a significant difference between tea grades/factories/elevations the Least Significant Difference (LSD) test is employed within the context of the analysis of variance. The R statistical software (version 3.2.3) was used to analyze the data.
3. Results and discussion
3.1. Different types of tea grades
Once the final product is obtained, the grading of tea is performed to achieve several objectives. One of these objectives is to meet trade requirements. Tea grading including its nomenclature, is based on the size of the leaves and the types of leaves included in the final product and this can vary with the type of tea and the country of origin. Zoysa et al. (2008) reported that different tea grades are produced at different tea-growing elevations in the country.
Based on the data collected during this study, it was observed that forty types of tea grades were produced in various tea factories in the country. These tea grades have been identified by registered tea brokers under the guidance of the Sri Lanka Tea Board. However, all these tea grades are not produced in every factory and there is also a lack of consistency in the production of different tea grades at three elevations. Fifteen major tea grades produced in each elevation is given in Table 2. Out of these Tea grades, some of the commonly found tea grades were Pekoe, Fannings, OP, OPA, FBOPF1, FBOP, etc.
Table 2.
Fifteen major tea grades produced in different elevations.
| Tea Grades | ||
|---|---|---|
| (Percentage of factories produced each tea grade) | ||
| Up country elevation | Mid country elevation | Low Country elevation |
| Fannings 1 (48%) | BOP1A (63%) | Pekoe (81%) |
| BOPF (41%) | FBOPF1 (58%) | FBOPF1(80%) |
| Dust1 (39%) | Pekoe (57%) | OP1 (77%) |
| Pekoe (35%) | FBOP (55%) | OPA (77%) |
| BOP (34%) | BP (55%) | FBOP (77%) |
| BPS (27%) | OPA (50%) | BOP1A (74%) |
| FBOP (26%) | BOPA (43%) | OP (70%) |
| Dust (25%) | Pekoe1 (41%) | Pekoe1 (68%) |
| FBOPF1 (23%) | OP1 (37%) | BOP1 (51%) |
| OPA (20%) | BOPF (36%) | BP (49%) |
| BOP1A (18%) | BOP1 (35%) | FBOPF (45%) |
| BOPsp (17%) | OP1 (37%) | FBOPFsp (44%) |
| BP (16%) | OP (35%) | Fannings (36%) |
| BOPA (15%) | FBOPF (33%) | BOPA (31%) |
| OP (15%) | Fannings (33%) | BOP (27%) |
BOP -Broken Orange Pekoe (Small/broken pieces of leaves).
BOP1A –Broken Orange Pekoe one A (Weight less large leaves).
BOP1-Broken Orange Pekoe one (Wiry and small than OP1).
BOPA -Broken Orange Pekoe (Smaller than a FBOP 1).
BOPF -Broken Orange Pekoe Fannings - Smaller than BOP leaves, broken leaf, slightly larger than dust.
BOPsp – Broken Orange Pekoe special (finest grade of tea which consists of neat smaller broken leaf without any other particles).
BP- Broken Pekoe.
BPS –Broken Pekoe Souchong.
D -Dust - Similar to D1 but will appear slightly brown powder leaves price must be low.
D1 -Dust 1 - The smallest of particles smaller than Fannings leaves.
Fannings- Small pieces of tea that are left over after higher grades of teas.
FBOP -Flowery Broken Orange Pekoe (Coarser and broken with some tips).
FBOP1- Flowery Broken Orange Pekoe one (firm and more black leaf with much better leafy tips).
FBOPF -Flowery Broken Orange Pekoe Fannings (Similar to the BOP leaf but firm leaf and with few tips).
FBOPF1 -Flowery Broken Orange Pekoe Fannings one (Similar to the BOPF but firm leaf and with little more tips then FBOPF).
FBOPFSP -Flowery Broken Orange Pekoe Fannings Special (Similar to the FBOPF1,firm and more black leaf with much better tips).
OP -Orange Pekoe (long wiry leaves without tips).
OP1 -Orange Pekoe One (long, wiry leaf with a light liquor).
OPA -Orange Pekoe ‘A’ (large and slightly open leaf pieces).
Pekoe -Twisted and Coarse.
Pekoe1 -Same style, but small in size than the Pekoe.
These results are similar to that of previous data reported by Zoysa et al. (2008) [30]. According to Zoysa et al. the prominent grades produced in up country elevation were Pekoe, BOPF, Fannings1, Dust1 and BOP whereas the major tea grades produced in mid country and low country elevations were Pekoe, BOP1, OP1, OP, OPA, BP, OPA, BOPA, Pekoe1, OP1, BOPF, BOP1, OP1, OP, and FBOPF. However, the results also revealed that two tea grades namely Silver tips and Golden tips were not in the list of the major fifteen tea grades produced in mid country and low country elevations and these are special grades produced by some selected tea factories.
3.2. Microbiological quality of graded black tea
Microbial load and the presence of specific microorganisms are accepted quality parameters in food products because these are related to the stability of the product. The variation of yeast and mould counts, APC and Coliform counts in different tea grades collected from different factories are shown in Fig. 2, Fig. 3. The aerobic plate counts and yeast and mould counts of 10 different tea grades were ranged from 1.5 × 103 to 1.9 × 104 cfu/g and 4.8 × 102 to 2.5 × 103 cfu/g respectively. Coliform counts were not detected in three different tea grades i.e GB, GD, GE and highest count was 3.9 × 101 MPN per g in GG graded tea.
Fig. 2.
Variation of aerobic plate counts and yeast & mould counts in different tea grades The alphabetical letters on the figure: means the mean values are significantly different at P < 0.05 n = GA (84), GB (63), GC (78), GD (66), GE (66), GF (45), GG (84), GH (75), GI (81), GJ (72).
Fig. 3.
Variation of coliform counts in different tea grades The alphabetical letters on the figure: means the mean values are significantly different at P < 0.05 n = GA (84), GB (63), GC (78), GD (66), GE (66), GF (45), GG (84), GH (75), GI (81), GJ (72).
Microbial counts of these tea grades were compared irrespective of the location of the factories, agro-climatic regions or elevations using a two-factor factorial randomized complete block design. The test results indicated that there was a significant difference in mean microbial counts among the tested tea grades (P < 0.05) (Fig. 2, Fig. 3).
The mean separation of microbial counts in tea grades using the Least Significant Difference (LSD) method indicated that the GG tea grade had significantly high counts (P < 0.05) of APC, yeasts & moulds and Coliforms when compared with other tea grades. The results also revealed, except for this tea grade, there was no significant difference in yeast & mould counts (Fig. 2) and coliforms counts (Fig. 3) among the remaining nine different tea grades. The comparison studies also revealed that three different tea grades i.e.GD, GC and GB have APC less than 3.0 × 103 cfu/g and these were significantly lower counts (P < 0.05) than other tea grades. Although high counts of yeast & mould and bacterial counts were found in some tea grades, E. coli and Salmonella spp. were absent in any of the tested tea grades. These two specific bacteria are considered as important quality parameters in food products.
The statistical analysis of the data also revealed that there was a significant difference (P < 0.05) in APC in different tea grades collected from different agro-climatic regions as well as different elevations, indicating that there is an effect from geographical positions of tea cultivation. Similar results were also observed with Coliform counts. In contrast to these results, the yeast and mould counts in these tea grades were not significantly different when compared with three different elevations, but it was significantly different (P < 0.05) with the agro-climatic regions.
Several studies have shown that there are different levels of microbiological contamination in tea. The tea samples collected from Sylhet and Moulvi bazaar area in Bangladesh have been reported to have a diverse number of total viable bacteria (TVB) counts ranging between 1.1 and 9.0 × 103 cfu/g and 1.1 × 103 cfu/g to 6.3 × 104 cfu/g. Coliform bacteria were detected in 20 % of samples [9]. However, generic E. coli and pathogenic E.coli 0157:H7 were detected in dried tea samples [31]. Anosike and Oranusi (2018) [32] also reported that bacterial counts ranged from 1.7 × 102 to 4.5 × 102 cfu/g in tea samples collected from the Nigerian retail market. A study was conducted to analyze 32 brands of (16 black teas and 16 green teas) commercially available tea in European and Italian markets [12]. They reported that microbial loads ranging from 1.0 × 102 to 2.8 × 105 cfu/g were observed in 80 % of the samples. A study conducted in India using black tea samples collected from 262 tea estates from 12 different districts of three major tea growing areas of Assam (Upper Assam, South Bank, North Bank) (Madhab et al., 2019) [14] showed that highest microbial contamination was present in the tea samples collected from South Bank in the summer season. E. coli, Salmonella and moulds were 7.0 × 102 cfu/g, 1.9 × 103 cfu/g and 8.5 × 103 cfu/g respectively. In accordance with the results, the highest microbial load has been observed as bacterial counts and followed by mould counts. According to them, the samples collected from Upper Assam contained the highest Salmonella counts whereas E. coli was not detected in the tea samples received from North bank [14].
When microbiological quality was assessed using the guidelines recommended by SLTB, more than 70 % of the tested samples of different tea grades were within the satisfactory microbiological quality (Table 3). An exception was observed with the tea grade-GG where approximately 60 % of the samples did not comply with the SLTB guidelines for yeast & mould counts and Coliform counts (Table 3). But, bacterial counts in 54 % of the tea grade-GG samples complied with the SLTB guidelines.
Table 3.
Percentage of tea samples complied with SLTB guideline.
| Tea Grade | Percentage of samples |
||
|---|---|---|---|
| YMC | APC | Coliforms | |
| GA | 93% | 100% | 89% |
| GH | 80% | 92% | 92% |
| GI | 74% | 85% | 96% |
| GC | 81% | 100% | 92% |
| GB | 80% | 100% | 100% |
| GD | 77% | 95% | 100% |
| GE | 95% | 100% | 100% |
| GF | 73% | 88% | 93% |
| GJ | 88% | 99% | 92% |
| GG | 39% | 54% | 36% |
| SLTB requirements | YMC <1000 CFU/g | APC <10000 CFU/g | Coliforms<10 MPN/g |
Number of samples analysed - GA (84), GB (63), GC (78), GD (66), GE (66), GF (45), GG (84), GH (75), GI (81), GJ (72).
It was observed that all the ten types of selected tea grades were not produced in every factory used in this study. However, the tea grades GH, GG, GI, GA and GC were produced in all the factories. The results revealed that over 85 % of the samples of GH, GI, GA, GC grades had Coliform counts less than 10 MPN/g (Table 3). According to SLTB regulatory requirements (SLTB Circular No: AL/MQS-02/2021) [33], yeast and mould counts and bacterial counts as APC should be < 1000 cfu/g and <10000 cfu/g, respectively. Approximately 75 % of the samples of GH, GC and GI met the SLTB requirements for both bacteria and yeast & mould counts. However, all the samples of GA, GC, GE and GB tea grades, had APC <10000 CFU/g which complied with the SLTB guidelines. Furthermore, Coliform counts in all the tested tea samples of GB, GD and GE have complied with the SLTB guideline requirements (Table 3).
3.3. Comparison of microbiological properties of tea produced in each tea factory with agro-climatic regions and elevations
It was assumed that the composite tea sample from each factory would represent the general microbiological quality of tea produced in the factory. The results were reported as variation of APC, yeast & mould and coliform counts in composite tea samples obtained from various factories located in the different agro-climatic regions (Table 4). The average APC and yeast & mould counts in composite tea samples collected from different factories ranged from 1.9 × 103 – 3.3 × 104 cfu/g and 3.0 × 102 to 6.0 × 103 cfu/g respectively (Table 4).
Table 4.
Variation of aerobic bacteria, yeast and mould and coliform counts in composite tea samples obtained from 28 factories located in seven different agro-climatic regions and the three elevations.
| Elevations | Agro-climatic region | Aerobic bacterial count (APC) CFU/g (log10) | Yeasts & Moulds count (YMC) CFU/g (log10) | Coliforms count |
|---|---|---|---|---|
| MPN/g (log10) | ||||
| Low country | Sabaragamuwa | 3.32–3.90 ± 2.79e | 2.57–3.78 ± 2.62a | 0.62–0.95 ± 0.26c |
| Ruhuna | 3.30–4.04 ± 2.58f | 2.48–3.43 ± 2.00d | ND – 2.08 ±0d | |
| Mid country | Kandy | 3.28–4.43 ± 3.23b | 2.73–3.11 ± 1.99c | ND -1.61 ± 0.51b |
| Up country | Dimbula | 3.78–4.52 ± 2.86a | 2.62–2.73 ± 2.26d | ND – 1.32 ± 0.32a |
| Nuwaraeliya | 3.60–4.04 ± 2.43c | 2.76–2.95 ± 1.68e | ND - 0.71 ± 0.04e | |
| Udapussallawa | 3.65–3.75 ± 3.08d | 2.48–3.28 ± 1.91b | ND – 1.26 ±0d | |
| Uva | 3.36–3.48 ± 3.78g | 2.57–2.81 ± 2.11f | ND – 0.71 ± 0.63e |
Number of samples analysed - Sabaragamuwa (117), Ruhunu (297), Kandy (93), Dimbula (42), Nuwaraeliya (72), Udapussallawa (39), Uva (54).
Data are represented as minimum and maximum values ± SE (n = 6). Different letters in a column superscripted means the mean values are significantly different at P < 0.05.
The yeast & mould counts in composite tea samples collected from the Sabaragamuwa region were higher than that of the other six agro-climatic zones (Table 4). The results also revealed that yeast and mould counts in tea samples collected from the factories located in Dimbula and Ruhunu agro-climatic regions were not significantly different from each other.
A significant difference was observed (P < 0.05) in the bacterial counts of composite tea samples collected from different agro-climatic zones. The APC counts were significantly high in the Dimbula region and counts were significantly low in Uva region (Table 4).
The dispersion of yeast and mould counts and the distribution of factories by agro-climatic regions and by the elevations are depicted in Fig. 4. When the interaction among agro-climatic zones and yeast and mould counts was considered it showed that the effect was significant (P < 0.05). Similar results were also observed between yeast & mould counts and the three different elevations (P < 0.05).
Fig. 4.
Dispersion of yeast and mould counts in composite tea samples collected from different factories in different agro climatic regions and three elevations n = Low country (414), Mid country (93), Up country (207).
The presence of yeast and mould contaminants in tea has special concern since black tea is generally a dried product with low water activity. The presence of fungi also can get affected by the different climatic conditions. However, the results indicated that the effect of the elevations on yeast and mould counts in tea samples was not significant (P < 0.05) but the effect of the agro-climatic regions on this group of organisms was significant (P < 0.05). The composition of phyllospheric microorganisms varies with geographical location influenced by factors such as climate variations and soil composition. The growth of phyllosphere microorganisms depends on the nitrogen and phosphorus contents which are in turn governed by the physical and chemical properties of the soil. The soil's microbial population serves as a crucial storage reservoir for phyllosphere microorganisms [34]. Therefore, it appears that identification of individual microorganisms present in tea grades collected from different elevations and/or agro-climatic regions might be useful to some extent to assess the variations in physical and chemical quality of the final tea grades.
The dispersion of Total Aerobic Plate Counts (i.e. APC) in composite tea samples obtained from different factories located in agro-climatic regions and elevations are shown in Fig. 5.
Fig. 5.
Dispersion of total aerobic bacterial counts in tea samples collected from different factories located in different agro climatic regions and three elevations n = Low country (414), Mid country (93), Up country (207).
The results revealed that APC counts were significantly higher (P < 0.05) in samples collected from Dimbula agro-climatic region compared to the counts in Sabaragamuwa, Ruhunu and Uva agro-climatic regions (Fig. 5).
The comparison studies revealed that the results of APC in tea samples obtained from the factories located in the Low country elevation were significantly lower (P < 0.05) than the other two elevations (Fig. 5). This may be due to the fact that in the low country elevation, the humidity is comparatively lower than in the other two elevations and the temperature is generally high. It was also observed that in the factories belonging to this elevation, the two parameters that can affect the growth of microorganisms i.e. temperature and humidity were maintained at similar values. Furthermore, the different steps in the production process were carried out within similar time intervals with slight deviations. This can delay the change of moisture content into higher levels in the final product. The moisture content together with high water activity can increase the growth of bacteria in food products.
Coliform counts were significantly higher (P < 0.05) in the Dimbula agro-region whereas Uva and Nuwaraeliya regions were not significantly different from each other (Fig. 6).
Fig. 6.
Total coliform counts of different factories in different agro climatic regions and three elevations. n = Low country (414), Mid country (93), Up country (207).
The analyses data also indicated that the interaction between different elevations and coliform counts was significantly higher in mid country elevation (P < 0.05) than that of the other two elevations But the values are not significantly different from each other in up country (5.0 ± 1.07) and low country (5.0 ± 0.81) elevations (Fig. 6).
The high coliform counts may be attributed to inadequate control of critical points. Additionally, coliforms may have survived the firing process and/or new organisms may have entered the manufacturing process after firing. Since coliforms are one of the microbiological indicators in food and water, the presence of this group generally indicates the presence of other enteric pathogens such as Salmonella spp. etc. During this study, it was evident that both Salmonella spp. and E. coli were not present in any of the tested tea samples.
3.4. Identification of isolated microorganisms
According to DNA characterization, bacteria in tea samples mainly belonged to two phyla i. e Firmicutes and Proteobacteria. Among the identified bacteria, 83 % belonged to Firmicutes family and 15 % were belonging to Proteobacteria.
In relation to fungal diversity, based on ITS sequences, 3 phyla namely Ascomycota, Basidiomycota and Zygomycota were identified. Among the identified moulds and yeasts 77 % belong to Ascomycota, 15 % belong to Basidiomycota and 8 % belong to Zygomycota. The results revealed that majority of the bacteria belongs to the genus Bacillus and majority of the fungal isolates belongs to the genus Aspergillus (Table 5). In previous studies, it was revealed that in the manufacturing process of black and green tea, the most common bacteria belong to the Firmicutes and Proteobacteria families while moulds and yeasts belong to the Ascomycota and Basidiomycota [12]. According to their results, the most commonly isolated bacteria were reported as Bacillus species with other isolated species identified as Paenibacillus spp., Pseudomonas, Staphylococcus and Clostridium. The reported moulds species were Aspergillus, Cryptococcus, Rhizopus, Mucor, Penicillium, Cladosporium, Cryptococcus and some identified bacteria belong to species of Cellulomonas, Corynebacterium and Shigella [11]. Tea sample contaminations have been reported with Aspergillus, Cladosporium, ramotenellum, Rhizopus, Rhodotorulamucillaginosa, Pantoeagavinia, Pseudomona, Salmonella spp. and Staphylococcus [12,35].
Table 5.
Identified bacteria, yeasts and moulds spp.
| Bacteria Spp. | Yeasts & Moulds Spp. | |
|---|---|---|
| Bacillus amyloliquefaciens Bacillus cereus Bacillus invictae Bacillus liqueniformis Bacillus pumilis Bacillus subtilus Bacillus safensis Bacillus thuringensis Enterobacter asburiae Kocuria kristinae Klebsiella variicola Lactobacillus fermentum Lysinibacillus macroides |
Aspergillus niger Aspergillus ochraceus Aspergillus tubingensis Aspergillus unguis Candida tropicalis Cladosporium cladosporioides Cladosporium gossypiicola Cladosporium oxysporum Cladosporium tenuissimum Debaryomyces hansenii |
Fusarium oxysporum Fusarium phaseoli Hyphopichia bartonii Kalmusia variispora Ophiocordyceps sinensis Penicillium oxalicum Peyronellaea glomerata Rhodosporidiobolus ruineniae Tricholoma matsutake Wickerhamomyces anomalus |
4. Conclusion
This investigation demonstrated that the assessed microbiological parameters i.e. E. coli and Salmonella were absent in any of the tea grades. This study also demonstrated that tea grade-GG had significantly high (P < 0.05) APC, yeast & moulds and Coliforms counts. The variations of microbial quality of tea produced in different factories located in different agro-climatic regions and elevations are statistically significant. The bacteria and the yeasts & moulds identified in the tea samples belong to the phyla Firmicutes (83 %), Proteobacteria (15 %), Ascomycota (77 %), Basidiomycota (15 %) and Zygomycota (8 %). Therefore, remedial actions must be implemented towards proper manufacturing of tea products and both quality assurance and quality control programs are required. The water quality using in the manufacturing process and microbial air quality in the factories should be monitored throughout the process.
Data availability statement
Data will be made available on request.
CRediT authorship contribution statement
S.H.S. Karunaratne: Writing – original draft, Visualization, Validation, Software, Resources, Project administration, Methodology, Investigation, Funding acquisition, Formal analysis, Data curation, Conceptualization. G.A.S.I. Abeygunawardena: Writing – review & editing, Supervision, Resources, Project administration, Methodology, Investigation, Data curation, Conceptualization. D.L. Jayaratne: Writing – review & editing, Supervision, Project administration, Methodology, Investigation. G.A.S. Premakumara: Writing – review & editing, Supervision, Resources, Project administration, Investigation, Funding acquisition, Data curation, Conceptualization.
Declaration of competing interest
The authors declare the following financial interests/personal relationships which may be considered as potential competing interests:S. H. S. Karunaratne reports financial support was provided by Sri Lanka Treasury. If there are other authors, they declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.
Acknowledgments
Sri Lanka Treasury for funding and Mr. N. Gunarathne (Agriculture sector modernization -World Bank funded project) for assistance in the statistical analysis.
References
- 1.Yao L.H., Jiang Y.M., Caffin N., D'Arcy B., Datta N., Liu X., Xu Y. Phenolic compounds in tea from Australian supermarkets. Food Chem. 2006;96(4):614–620. doi: 10.1016/j.foodchem.2005.03. [DOI] [Google Scholar]
- 2.Jayasekera S., Molan A.L., Garg M., Moughan P.J. Variation in antioxidant potential and total polyphenol content of fresh and fully-fermented Sri Lankan tea. Food Chem. 2011;125(2):536–541. doi: 10.1016/j.foodchem.2010.0. [DOI] [Google Scholar]
- 3.Von Staszewski M., Jagus R.J., Pilosof A.M.R. Influence of green tea polyphenols on the colloidal stability and gelation of WPC. Food Hydrocolloids. 2011;25(5):1077–1084. doi: 10.1016/j.foodhyd.2010.10.0. [DOI] [Google Scholar]
- 4.Saito S.T., Gosmann G., Saffi J., Presser M., Richter M.F., Bergold A.M. Characterization of the constituents and antioxidant activity of Brazilian green tea (Camellia sinensis var. assamica IAC-259 cultivar) extracts. J. Agric. Food Chem. 2007;55(23):9409–9414. doi: 10.1021/jf071796p. [DOI] [PubMed] [Google Scholar]; [5a] Kim Y., Goodner K.L., ParK J.D., Choi J., Talcott S.T. Changes in antioxidant phytochemicals and volatile composition of Camellia sinensis by oxidation during tea fermentation. Food Chem. 2011;129(4):1331–1342. doi: 10.1016/j.foodchem.2011.05.012. [DOI] [Google Scholar]; [5b] Kim J.Y., Park J., Park Y. Analysis of microflora in gochujang, Korean traditional fermented food. Food Sci. Biotechnol. 2011;20(5):1435–1440. [Google Scholar]
- 5.Xu A.Q., Wang Y.L., Wen J.Y., Liu P., Liu Z.Y., Li Z.J. Fungal community associated with fermentation and storage of Fuzhuan brick-tea. Int. J. Food Microbiol. 2011;146(1):14–22. doi: 10.1016/j.ijfoodmicro.2011. [DOI] [PubMed] [Google Scholar]
- 6.Dharmadasa M., Zubair L., Nijamdeen L., Najimuddi N. Review of tea industry in Sri Lanka for climate analysis. Tropical climate. 2018 www.tropicalclimate.org [Google Scholar]
- 7.Jayasinghe S.L., Kumar L. Potential impact of the current and future climate on the yield, quality and climate suitabllity for tea (Camellia sinensis (L.) O.Kuntze): a systematic review. Agronomy. 2021;11:619. [Google Scholar]
- 8.Elshafie A.E., Al-Lawatia T., Al-Bahry S. Fungi associated with black tea and tea quality in the Sultanate of Oman. Mycopathologia. 1999;145(2):89–93. doi: 10.1023/a:1007034729467. [DOI] [PubMed] [Google Scholar]
- 9.Rashid M.H., Chowdhury M.A.Z., Fardous Z., Tanvir E.M., Pramanik M.K., Jahan I., Alam K., Moniruzzaman M., Gan S.H. Microbial decontamination of gamma irradiated black tea and determination of major minerals in black tea, fresh tea leaves and tea garden soil. Food Sci. Technol. 2016;73:185–190. [Google Scholar]
- 10.Mishra B., Gautam S., Sharma A. Microbial decontamination of tea (Camellia sinensis) by gamma radiation. J. Food Sci. 2006;71(6):151–156. [Google Scholar]
- 11.Ossowski M., Nowakowicz-Debek B., Wlazlo L., Krol J., Kasela M., Maksym P., Malm A. Evaluation of microbiological contamination of black and green teas. Adv. J. Food Sci. Technol. 2019;17(4):65–71. doi: 10.19026/ajfst.17.6016. [DOI] [Google Scholar]
- 12.Carraturo F., De Castro O., Troisi J., De Luca A., Masucci A., Cennamo P., Trifuoggi M., Aliberti F., Guida M. Comparative assessment of the quality of commercial black and green tea using microbiology analyses. BMC Microbiol. 2018;18:4. doi: 10.1186/s12866-017-1142-z. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 13.Dharmarathnaa E.K.G.P.U., Liyanawickramasinghea T.R., Aruppalab A.L.Y.H., Abeyrathne E.D.N.S. A study on level of microbiological contamination in made tea as a raw material in commercial tea bagging factory and its workers' hand hygiene. Journal of Agriculture and Value Addition. 2018;1(2):49–59. [Google Scholar]
- 14.Madab M., Bhattacharyya P.N., Begum R., Dutta P., Tanti A.J., Konwar M., Hazarika S., Sarmah S. Microbial distribution and contamination in black tea. Two Bud. 2019;64(1):26–31. [Google Scholar]; [17a] Owuor P.O., Obaga S.O., Othieno C.O. Effects of altitude on the chemical composition of black tea. J. Sci. Food Agric. 1990;50:9–17. [Google Scholar]; [17b] Owuor P.O., Obanda M., Nyirenda M.H., Mandala W.L. Influence of region of production on clonal black tea chemical characteristics. Food Chem. 2008;108:263–271. [Google Scholar]
- 15.Magoma G., Wachira F.N., Obanda M., Imbuga M., Agong S.G. The use of catechins as biochemical markers in diversity studies of tea (Camellia sinensis) Genet. Resour. Crop Evol. 2000;47:107–114. [Google Scholar]
- 16.Tea Exporters Association in Sri Lanka Tea grade nomenclature. 2017. https://teasrilanka.org/tea-grade
- 17.International Organization for Standardization . 2017. Microbiology of the Food Chain - Preparation of Test Samples, Initial Suspension and Decimal Dilutions for Microbiological Examination - Part 1: General Rules for the Preparation of the Initial Suspension and Decimal Dilutions. ISO 6887-1. [Google Scholar]
- 18.International Organization for Standardization Microbiology of food and animal feeding stuffs - Horizontal method for the enumeration of yeasts and moulds - Part 2: Colony count technique in products with water activity less than or equal to 0.95. 2008 ISO 21527-2. [Google Scholar]
- 19.International Organization for Standardization . 2013. Microbiology of the Food Chain-Horizontal Method for the Enumeration of Microorganisms - Part 1: Colony Count at 30 °C by the Pour Plate Technique. ISO 4833-1. [Google Scholar]
- 20.International Organization for Standardization . 2006. Microbiology of Food and Animal Feeding Stuffs - Horizontal Method for the Detection and Enumeration of Coliforms -Most Probable Number Technique. ISO 4831. [Google Scholar]
- 21.International Organization for Standardization . 2005. Microbiology of Food and Animal Feeding Stuffs - Horizontal Method for the Detection and Enumeration of Presumptive Escherichia coli - Most Probable Number Technique. ISO 7251. [Google Scholar]
- 22.International Organization for Standardization . 2017. Microbiology of the Food Chain - Horizontal Method for the Detection, Enumeration and Serotyping of Salmonella - Part 1: Detection of Salmonella Spp. ISO 6579-1. [Google Scholar]
- 23.Sneath P.H.A., Mair N.S., Sharpe M.E., Holt J.G. Bergey’s manual of Systematic bacteriology. 1986;2 [Google Scholar]
- 24.Hongoh Y., Ohkuma M., Kudo T. Molecular analysis of bacterial microbiota in the gut of the termite Reticulitermes speratus (Isoptera; Rhinotermitidae) FEMS (Fed. Eur. Microbiol. Soc.) Microbiol. Ecol. 2003;1495(1):12. doi: 10.1016/S0168-6496(03)00026-6. [DOI] [PubMed] [Google Scholar]
- 25.Sung J.Y., Hwang Y., Shin M.H., Park M.S., Lee S.H., Yong D., Lee K. Utility of conventional culture and MALDI-TOF MS for identification of microbial communities in bronchoalveolar lavage fluid in comparison with the GS junior next generation sequencing system. Annals of Laboratory Medicine. 2018;38:110–118. doi: 10.3343/alm.2018.38.2.110. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 26.Wang X., Fu Y.-F., Wang R.-Y., Li L., Cao Y.-H., et al. Identification of clinically relevant fungi and prototheca species by rRNA gene sequencing and multilocus PCR coupled with electrospray ionization mass spectrometry. PLoS One. 2014;9(5) doi: 10.1371/journal.pone.0098110. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 27.Raja H.A., Miller A.N., Pearce C.J., Oberlies N.H. Fungal identification using molecular tools: a primer for the natural products research community. J. Nat. Prod. 2017;80:756–770. doi: 10.1021/acs.jnatprod.6b01085. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 28.Alsohaili S.A., Bani-Hasan B.M. Morphological and molecular identification of fungi isolated from different environmental sources in the northern eastern desert of Jordan. Jordan J. Biol. Sci. 2018;11:329–337. [Google Scholar]
- 29.Javadi M.A., Ghanbary M.A.T., Tazick Z. Isolation and molecular identification of soil inhabitant Penicillia. Ann. Biol. Res. 2012;3(12):5758–5761. [Google Scholar]
- 30.Zoysa A.K.N., Sivapalan P., Kulasegaram S., Kathiravetpillai A. 2008. Hand Book on Tea. [Google Scholar]
- 31.Zaman S., Alam M.K., Ahmed S., Uddin M.N., Bari M.L. The Prevalence of E. coli O157:H7 in the production of organic herbs and a case study of organic lemongrass intended for use in blended tea. Agriculture. Food and Analytical Bacteriology. 2014;4(3):164–176. [Google Scholar]
- 32.Anosike S.O., Oranusi S. Assessment of microbiological and chemical qualities of selected cocoa, tea and coffee brands in Nigerian markets. Afr. J. Clin. Exp. Microbiol. 2018;19(3) doi: 10.4314/ajcem.v19i3.5. [DOI] [Google Scholar]
- 33.Sri Lanka tea board minimum quality standards for tea. SLTB – D (AS), SLTB-MQS 02/SL, AL/MQS-02/2021- Annexure A. 2021 [Google Scholar]
- 34.Huang S., Zha X., Fu G. Affecting factors of plant phyllosphere microbial community and their responses to climatic warming -A review. Plants. 2023;12:2891. doi: 10.3390/plants12162891. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 35.Bouakline A., Lacroix C., Roux N., Gangneux J.P., Derouin F. Fungal contamination of food in hematology units. J. Clin. Microbiol. 2000;38(11):4272–4273. doi: 10.1128/JCM.38.11.4272-4273.2000. [DOI] [PMC free article] [PubMed] [Google Scholar]
Associated Data
This section collects any data citations, data availability statements, or supplementary materials included in this article.
Data Availability Statement
Data will be made available on request.