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Ruthenium (II) complexes interact with human serum albumin and induce apoptosis of tumor cells.
Dairy Sci. & Technol. (2014) 94:327–339
DOI 10.1007/s13594-014-0165-6
O R I G I N A L PA P E R
Effect of green tea supplementation
on the microbiological, antioxidant, and sensory
properties of probiotic milks
Dorota Najgebauer-Lejko
Received: 5 August 2013 / Revised: 12 February 2014 / Accepted: 13 February 2014 /
Published online: 26 March 2014
# The Author(s) 2014. This article is published with open access at Springerlink.com
Abstract Green tea and its constituents are known for a wide range of healthpromoting properties. They may exert antimicrobial action but without altering
lactic acid bacteria. The aim of the present study was to estimate the effect of
green tea addition on the selected properties of probiotic milks. Bioyogurts
(fermented with ABT-1 coculture of Streptococcus thermophilus, Lactobacillus
acidophilus LA-5, Bifidobacterium animalis subsp. lactis BB-12) and acidophilus
milks (fermented with pure L. acidophilus LA-5 culture) with addition of 0, 5,
10, or 15% (v/v) green tea infusion (GTI) were produced and analyzed for the
antioxidant capacity by the “diphenyl picrylhydrazyl” (DPPH) and “ferric-reducing antioxidant power” (FRAP) methods, acidity, the count of starter bacteria,
and sensory properties at the 1st, 7th, 14th, and 21st day of cold storage. The
15% addition of GTI to the acidophilus milk significantly reduced the lactic acid
production during the whole study. The GTI had no impact on the level of
S. thermophilus and B. lactis BB-12 in bioyogurts, and its effect on the count of
L. acidophilus LA-5 depended on the concentration and probiotic milk type. GTI
similarly and in a dose-dependent manner enhanced the antioxidant capacity of
both milk types. There were no significant differences between the sensory notes
received for bioyogurts, whereas acidophilus milks with tea were less appreciated
by the panelists. In conclusion, green tea could be successfully used as a
functional additive for selected probiotic milks enhancing their health benefits,
but the proper selection of tea additive and starter culture is recommended.
Keywords Green tea . Probiotics . Fermented milk . Antioxidant activity . Sensory
evaluation
D. Najgebauer-Lejko (*)
Faculty of Food Technology, Department of Animal Product Technology,
University of Agriculture in Krakow, ul. Balicka 122, 30-149 Krakow, Poland
e-mail: d.najgebauer-lejko@ur.krakow.pl
328
D. Najgebauer-Lejko
1 Introduction
Probiotics are defined as “live microorganisms which, when administered in
adequate amounts, confer a health benefit on the host” (FAO/WHO 2006).
Fermented dairy products, such as yogurt, are very popular food delivery systems
of live probiotic cells. In recent years, due to various therapeutic benefits documented and increased consumer health awareness, the popularity of the functional
dairy products containing probiotics has significantly increased. The health benefits linked with the consumption of probiotic microorganisms most commonly
used in dairy products, i.e., belonging to Lactobacillus and Bifidobacterium
genera, comprise prevention and relieving effects in various types of diarrhea
(infantile, traveler’s, antibiotic-associated), alleviation of gastrointestinal complaints, reduction of lactose intolerance, lowering serum cholesterol level, anticarcinogenic activity, prevention of urogenital infections, reduction of allergic symptoms, stimulation of the immune system, etc. (FAO/WHO 2006; Sanders et al.
2013). It is important that probiotic food products must contain living probiotic
strains in an adequate matrix and in sufficient concentration at the time of
consumption to reach after ingestion the postulated effect. However, the main
problems connected with incorporating probiotic bacteria into fermented milk
formulae are their slow growth in milk and loss of viability during storage (ElDieb et al. 2012). Different strategies can be applied to support the growth of
probiotic bacteria in milk, e.g., microencapsulation of probiotic cells, heat shock of
the yogurt before addition of probiotics, proper selection of starter cultures,
addition of prebiotic substances (e.g., inulin, oligosaccharides), etc. (El-Dieb
et al. 2012; Oliveira et al. 2012). Dave and Shah (1997) used ascorbic acid
additive as oxygen scavenger to make the environment more conducive, i.e., with
reduced oxygen content and redox potential, for these microaerophilic
(Lactobacillus acidophilus) or strictly anaerobic (Bifidobacterium ssp.) microorganisms. Probiotic milks are also often supplemented with other active components with the aim to provide additional functional properties, like plant sterols
and stanol esters as well as antioxidative substances (Saxelin 2008).
Tea is the second, next to water, most commonly consumed beverage worldwide. Many studies have been conducted which demonstrated beneficial effects of
tea and its constituents on human health. The health claims include reduction of
risk of cancer, arteriosclerosis and cardiovascular diseases, neural and obesity
problems, diabetes, pulmonary ailments, and diseases of the kidneys and liver,
and antibacterial and antiviral effects. The most important bioactive substances
responsible for these health-giving properties present in tea are flavonoids (namely,
catechins and their derivatives) (Jain et al. 2006). Among all functions of polyphenols, their antioxidant activity is the most frequently studied. Their in vitro
action as antioxidants refers to the scavenging activity against reactive oxygen and
nitrogen species and the ability to sequester metal ions (Bancirova 2010; Zhu et al.
2006). Polyphenols have been also reported as very potent antimicrobial agents
(von Staszewski et al. 2011). The study of Almajano et al. (2008) indicated that
tea compounds characterized by the highest antioxidant power are simultaneously
the most effective as microbiological inhibitors. Phenolics present in tea and wine
are able to modify the intestinal microbiota by inhibiting the growth of pathogenic
Probiotic milks with green tea
329
bacteria and increasing the level of commensal bacteria, including bifidobacteria,
which suggests their prebiotic effect (Hara 1997; Lee et al. 2006; Queipo-Ortuño
et al. 2012). Catechins from tea and other phenolic compounds have also been
shown to inhibit the growth of many food-borne bacteria and fungi in milk with
little effect on lactic acid bacteria (Almajano et al. 2008; O’Connell and Fox
2001). The sensitivity of lactic acid bacteria (LAB) and bifidobacteria to the
phenolic compounds depends on the bacterial species and strain, as well as
chemical structure and concentration of the polyphenols (Tabasco et al. 2011).
Some of the resistant strains, e.g., Lactobacillus plantarum, Lactobacillus casei
Shirota, are also able to metabolize these compounds (Lee et al. 2006; Rodríguez
et al. 2009).
In addition to their benefits for human health, phenolics also affect sensory properties, i.e., flavor, taste (astringency), and color of the food products (Rodríguez et al.
2009). These facts make tea not only very popular as a beverage, but its extracts are
also successfully incorporated into food systems, e.g., ice cream mixes, yogurt, and
fruit-flavored milk drinks (O’Connell and Fox 2001).
Previous studies demonstrated that addition of tea extracts to the conventional
yogurt, i.e., containing Streptococcus thermophilus and Lactobacillus delbrueckii
subsp. bulgaricus, did not affect the viability of starter microorganisms (Jaziri et al.
2009) or the stimulating effect was observed (Najgebauer-Lejko et al. 2011). However,
there is lack of information on the effect of tea in fermented milks containing probiotic
bacteria. This study was established to estimate the effect of green tea infusion (GTI)
supplementation in different concentrations (0, 5, 10, or 15% v/v) on the antioxidant
capacity measured as scavenging activity against DPPH radical and ferric-reducing
ability (FRAP) of bioyogurts (milks fermented with the ABT-1 coculture of
S. thermophilus and two probiotic strains, i.e., L. acidophilus LA-5 and
Bifidobacterium animalis ssp. lactis BB-12) and acidophilus milks (fermented with
pure L. acidophilus LA-5 culture) and to determine whether GTI can affect viability of
starter cultures, including probiotic bacteria, during a 3-week cold storage. Moreover,
the acidity and sensory quality of the plain and supplemented probiotic milks were
evaluated.
2 Materials and methods
2.1 Materials
Fresh, raw, cows’ milk (∼20 L) for the production of probiotic milks was obtained from
a local milk farm in Dziekanowice (Poland). Chinese leaf green tea (Yunnan Tea
Garden Group Shareholding Co., Ltd., Kunming, China) was purchased from the local
supermarket. Instant nonfat milk powder was purchased from Dairy Company in
Gostyn (Poland). ABT-1 DVS coculture, consisting of S. thermophilus,
L. acidophilus (LA-5), and B. lactis (BB-12), and L. acidophilus LA-5 monoculture
were obtained from Chr. Hansen (Hoersholm, Denmark).
Folin-Ciocalteu’s phenol reagent, Trolox, and gallic acid monohydrate were purchased from Fluka (Buchs, Switzerland; Copenhagen, Denmark; and Madrid, Spain),
and 2,2-diphenyl-1-picrylhydrazyl (DPPH) and 2,4,6-tris(2-pyridyl)-s-triazine (TPTZ)
330
D. Najgebauer-Lejko
from Sigma-Aldrich (Steinheim, Germany and Buchs, Switzerland). All other
chemicals used were of analytical reagent grade.
2.2 Preparation of tea infusion
Green tea leaves (40 g) were infused in 800 mL of freshly boiled water in a glasscovered beaker for 15 min. The leaves were then removed and the infusion cooled
to ambient temperature. To achieve the same content of milk solids in all final
products, a boiled-and-cooled-to-ambient-temperature water in the proper amounts
was added to milk destined for the nonsupplemented (natural) probiotic milks
(bioyogurt and acidophilus milk) as well as for the milks with 5 or 10% (v/v) of
tea infusions.
2.3 Manufacturing of bioyogurts and acidophilus milks
The preparation of milk for probiotic milk products comprised centrifugation (3,500×g,
45 °C) to reach 2% fat level, standardization with nonfat milk powder (NMP) to
achieve 15% dry matter content in the final products, homogenization (60 °C,
6 MPa), pasteurization (85 °C, 15 min), and cooling to 38 °C. Subsequently, the bulk
milk was inoculated with the ABT-1 starter (0.08 g per 1 L of milk) for bioyogurts or
LA-5 culture (0.1 g per 1 L of milk) for acidophilus milks. Each treatment was divided
into four equal portions; mixed with proper amounts of GTI or/and water to reach 0, 5,
10, or 15% of GTI; and poured into 200-mL sterile glass jars. The incubation proceeded
at 37 °C for 10–12 h (the same time for all milks) until firm coagula were formed (pH
of 4.6–4.8 for all treatments except for acidophilus milk with 15% of GTI which was
higher). Subsequently, fermented milk products were immediately cooled and stored at
4 °C prior to analyses. The samples were subjected to analyses directly after production
and after 7, 14, and 21 days of refrigerated storage at ∼4 °C.
2.4 Analyses of tea infusion
Green tea infusion was analyzed for the total level of polyphenolic compounds using the
Folin-Ciocalteu method by the procedure described by Rusak et al. (2008). Results
expressed as milligrams of gallic acid equivalents (GAE) per liter of infusion were
recalculated per 100 g of the respective milk. Additionally, the content of flavan-3-ols,
i.e., (−)-epicatechin (EC), (−)-epigallocatechin (EGC), (−)-epigallocatechin gallate
(EGCG), and (−)-epicatechin gallate (ECG), was determined by means of highperformance liquid chromatography (HPLC). The catechins were identified using
L-7000 LaChrom liquid chromatograph with UV/Vis detector (Merck-Hitachi, Tokyo,
Japan) equipped with a reversed phase (ODS) column (Thermo Scientific, USA;
25 mm×0.4 cm×5 μm). The sample preparation and exact analysis followed the
procedure described by Socha et al. (2013). The determination of ferric-reducing antioxidant power (FRAP) and scavenging rate of 2,2-diphenyl-1-picrylhydrazyl (DPPH)
radical followed the procedures described in the previous work of Najgebauer-Lejko
et al. (2011). The FRAP was expressed as millimoles Fe2+ equivalents (E) per liter, and
the DPPH antiradical activity as antiradical power (ARP) calculated relative to the ARP
of Trolox in millimoles of Trolox equivalents (TE) per kilogram sample.
Probiotic milks with green tea
331
2.5 Analyses of fermented milks
In all samples, pH was measured using an Elmetron (Zabrze, Poland) CP-411 pH meter.
Titratable acidity expressed as percentage of lactic acid was determined according to
the Soxhlet-Henkel method (Polish Standard, PN-A-86061:2002).
The level of S. thermophilus in bioyogurts was assessed using M17 agar
(Biocorp, Warszawa, Poland) under aerobic conditions. For enumeration of
L. acidophilus, MRS-maltose agar was prepared from the same ingredients and
procedure as MRS agar (ISO 7889:2003) but by replacing glucose with an equal
amount of maltose and adjusting pH to 6.4. The Petri dishes for lactobacilli
enumeration in bioyogurts and acidophilus milks were incubated respectively
under aerobic conditions or in 2.5-L anaerobic jars using Anaerocult® C sachets
(Merck, Darmstadt, Germany). The count of bifidobacteria was evaluated using
nalidixic acid, neomycin sulfate, lithium chloride, and paromomycin sulfate
(NNLP)-MRS agar (MRS supplied by Biocorp, Warszawa, Poland) in anaerobic
incubators of our own construction filled with CO2. All cultures were incubated
at 37 °C for 2 (streptococci) or 3 (lactobacilli, bifidobacteria) days.
The evaluation of the antioxidant capacity was also performed using DPPH and
FRAP methods by the same procedures as for the tea infusion. The sensory evaluation
was performed according to the PN-ISO 6658 standard (1998) using a 5-point scale
(from 5—excellent to 1—very bad) by a trained panel of five judges. The fermented
milk samples were presented to panelists in identical plastic cups labeled with random
numbers and in random order. The following properties were assessed: color, taste,
odor, consistency, and general appearance. The overall preference was calculated taking
into account the proper indexes of importance for each quality attribute (0.10, 0.35,
0.15, 0.25, 0.15, respectively).
2.6 Statistical analysis
All experiments were performed in three series and in duplicate and results expressed
as mean±standard error. Estimation of the effect of tea addition and time of storage was
conducted using ANOVA, and where the significant effect was found, the significance
of differences between the means was determined on the basis of Duncan’s test at the
significance level of P≤0.05. The statistical analysis was performed using Statistica 8.0
software (StatSoft Inc., Tulsa, OK, USA).
3 Results
3.1 Composition and acidity of fermented milks
The bioyogurts and acidophilus milks produced in this study contained on average
15.2% of dry matter (dm), 1.7% of fat, and 4.8% of protein as well as 0, 0.07, 0.15, or
0.22 g of tea dry matter per 100 g of fermented milk, respectively, for each level of
supplementation (data not shown). The addition of GTI to the examined probiotic milks
resulted in the enrichment of their composition with tea polyphenols in the amount of
57.9, 115.8, and 173.8 mg GAE per 100 g of fermented milk (for 5, 10, or 15% of tea
332
D. Najgebauer-Lejko
supplementation, respectively), as determined by the Folin-Ciocalteu method. HPLC
analysis revealed that the green tea infusion used for milk supplementation contained
81.25±0.23 (−)-epicatechin (EC), 148.47±0.24 (−)-epicatechin gallate (ECG), 147.36
±4.78 (−)-epigallocatechin (EGC), and 242.86±1.90 (−)-epigallocatechin gallate
(EGCG) (mg⋅100 mL−1 of tea infusion).
Directly after production, it could be observed that the pH of the bioyogurts and
acidophilus milks was slightly higher for higher levels of green tea supplementation,
which was accompanied by the lower values of titratable acidity (Table 1). This
phenomenon was much more pronounced in the case of milk products fermented
solely with L. acidophilus LA-5 showing the noticeable difference of 0.51 units and
0.42% lactic acid, respectively, for initial pH and titratable acidity noted between plain
acidophilus milk (NA) and that with 15% of green tea infusion (GTA-15%). The
significant difference in acidity of NA and GTA-15% was also observed after storage.
Generally, the pH values decreased and the level of lactic acid slightly increased
throughout the storage, although observed pH reduction was statistically significant
(P≤0.05) only for acidophilus milk with 15% of green tea. The drop in pH value
observed for this treatment was above twice that evaluated for the other milks
fermented with L. acidophilus LA-5 monoculture.
Table 1 Changes of the pH, titratable acidity, and sensory notes of bioyogurts and acidophilus milks during
cold storage (n=6)
Sample NY
GTY-5% GTY-10% GTY-15% NA
GTA-5% GTA-10% GTA-15% SE
Day
pH
1
4.54 a
4.61 a
4.70 ab
4.78 ab
4.51 a
4.62 a
4.69 ab
5.02 bA
0.05
7
4.54 ab 4.52 ab
4.58 ab
4.70 ab
4.38 a
4.48 ab
4.59 ab
4.83 b
0.05
14
4.50 ab 4.48 ab
4.60 ab
4.64 ab
4.32 a
4.48 ab
4.60 ab
4.74 b
0.04
21
4.48 a
4.50 a
4.65 a
4.34 a
4.45 a
4.59 a
4.64 aB
0.03
Day
4.50 a
Titratable acidity (% lactic acid)
1
1.07 ab 0.91 ab
0.87 ab
0.70 a
1.16 b
1.07 ab
0.95 ab
0.74 a
0.05
7
1.13 ab 1.12 ab
1.08 ab
0.75 a
1.25 b
1.22 b
1.09 ab
0.94 ab
0.05
14
1.12 ab 1.14 ab
0.96 ab
0.81 a
1.31 b
1.08 ab
1.00 ab
0.92 ab
0.05
21
1.13 ac
0.94 ac
0.81 a
1.31 c
1.24 bc
1.11 ac
0.90 ab
0.04
Day
1.08 ac
Sensory evaluation (scores)
1
4.77 a
4.72 a
4.78 a
4.70 a
4.53 a
4.19 a
4.20 a
4.17 a
0.07
7
4.78 c
4.69 bc
4.79 c
4.62 bc
4.04 ab
3.81 a
3.71 a
3.52 a
0.12
14
4.86 d
4.82 d
4.79 d
4.71 cd
4.43 bcd 4.07 ac
3.83 ab
3.67 a
0.10
21
4.66 b
4.60 b
4.48 b
4.43 b
4.20 ab
3.68 a
4.14 ab
0.10
3.99 ab
Means within each row not sharing the same lowercase letter are statistically different (P≤0.05); different
capital letters given in columns denote the statistical difference (P≤0.05) between means for a given feature
NY natural (plain) bioyogurt; GTY-5%, GTY-10%, and GTY-15%—bioyogurts with 5, 10, and 15% (v/v) of
green tea infusion, respectively; NA natural (plain) acidophilus milk; GTA-5%, GTA-10%, and GTA-15%—
acidophilus milks with 5, 10, and 15% of green tea infusion, respectively; SE standard error of the mean
Probiotic milks with green tea
333
3.2 Antioxidant capacity of probiotic milks as affected by green tea supplementation
Two methods were employed for measuring antioxidant activity of fermented milks,
i.e., DPPH assay, which measures the scavenging activity of the DPPH radical by
antioxidant substances present in the examined sample, and FRAP, which allows to
estimate the ability to reduce prooxidant metal ions. The results of FRAP evaluation
expressed as millimoles Fe2+ E per liter and DPPH analysis given as ARP in millimoles
of Trolox equivalents (TE) per kilogram sample are presented in Figs. 1 and 2,
respectively.
In the present study, ARP values evaluated for the plain bioyogurt (NY) and
acidophilus milk (NA) fluctuated in the range of 0.21–0.28 mmol TE kg−1 with no
significant differences between these two types of fermented milks (P>0.05). The
FRAP values were slightly higher for NA (1.25–1.61 mmol Fe2+ E L−1) than those
for NY (0.89–1.05 mmol Fe2+ E L−1), but also in this case, the differences were
insignificant. Green tea infusion was characterized by noticeably higher antioxidant
capacity with 41.60±0.73 mmol TE kg−1 and 72.76±6.74 mmol Fe2+ E L−1 average
ARP and FRAP values, respectively (data not shown).
The strong antioxidative properties of green tea resulted in significantly higher ARP
(9–29-fold) and FRAP (3–13-fold) values of all supplemented fermented milks when
16.00
a
FRAP (mmol Fe
2+
E L-1)
14.00
12.00
10.00
8.00
6.00
4.00
2.00
0.00
1
7
14
21
storage time (days)
FRAP (mmol Fe 2+ E L-1)
16.00
b
14.00
12.00
10.00
8.00
6.00
4.00
2.00
0.00
1
7
14
21
storage time (days)
Fig. 1 Changes of the ferric-reducing antioxidant power (FRAP) values of bioyogurts (a) and acidophilus
bioyogurt/acidophilus milk
milks (b) during refrigerated storage ( natural bioyogurt/acidophilus milk;
bioyogurt/acidophilus milk with 10% of green tea infusion;
bioyogurt/
with 5% of green tea infusion;
acidophilus milk with 15% of green tea infusion; means±SE, n=6)
□
334
D. Najgebauer-Lejko
7.00
a
-1
ARP (mmol TE k g )
6.00
5.00
4.00
3.00
2.00
1.00
0.00
1
7
14
21
storage time (days)
7.00
b
-1
ARP (mmol TE k g )
6.00
5.00
4.00
3.00
2.00
1.00
0.00
1
7
14
21
storage time (days)
Fig. 2 Changes of the antiradical power (ARP) values of bioyogurts (a) and acidophilus milks (b) during
bioyogurt/acidophilus milk with 5% of green
refrigerated storage ( natural bioyogurt/acidophilus milk;
bioyogurt/acidophilus milk with 10% of green tea infusion;
bioyogurt/acidophilus milk
tea infusion;
with 15% of green tea infusion; means±SE, n=6)
□
compared with the natural treatments. The higher level of tea additive was applied, the
higher radical-scavenging ability and ferric-reducing activity was observed, with statistically significant differences between the respective results (P≤0.05). There were no
differences between bioyogurts and acidophilus milks with the same level of supplementation. For all products, the FRAP and ARP values determined after 3 weeks of
storage were lower than the initial by 2–22% and 2–16% (respectively), but the rate of
this decrease was statistically insignificant.
3.3 Effect of green tea addition on the viability of the starter bacteria
The effect of green tea addition to the bioyogurts and acidophilus milks on the number
of starter microorganisms is shown in Fig. 3a–d. As regards probiotic strains,
lactobacilli were present in the number of 7.21–8.29 log cfu g−1 (bioyogurts) or
8.72–9.02 log cfu g−1 (acidophilus milks) and the count of bifidobacteria in
bioyogurts reached the level of 6.66–7.54 log cfu g−1. The levels of all starter
bacteria in both types of probiotic milks and in all treatments remained practically
unchanged after 3 weeks of refrigerated storage (no significant differences at P≤0.05).
S. thermophilus with the number of 8.47–9.12 log cfu g−1 was the prevailing species
in bioyogurts constituting from 69 to 95% (depending on storage duration and level of
Probiotic milks with green tea
b
9.5
8.5
8
9
log cfu g-1
log cfu g-1
a
335
8.5
7.5
7
6.5
6
8
1
NY
7
GTY-5%
GTY-10%
14
GTY-15%
1
21
storage time (days)
c 8.5
NY
GTY-10%
GTA-5%
GTA-10%
14
GTY-15%
21
storage time (days)
d
9.1
8
9
log cfu g-1
log cfu g-1
7
GTY-5%
7.5
7
6.5
8.9
8.8
8.7
8.6
6
8.5
1
NY
7
GTY-5%
GTY-10%
14
GTY-15%
21
storage time (days)
1
NA
7
14
GTA-15%
21
storage time (days)
Fig. 3 Viability of starter bacteria in probiotic milks with or without green tea additive during refrigerated
storage: a Streptococcus thermophilus count in bioyogurts, b Bifidobacterium animalis ssp. lactis count in
bioyogurts, c Lactobacillus acidophilus count in bioyogurts, and d Lactobacillus acidophilus count in
acidophilus milks. NY natural bioyogurt; GTY-5%, GTY-10%, and GTY-15%—bioyogurt with 5, 10, and
15% of green tea infusion, respectively; NA natural acidophilus milk; GTA-5%, GTA-10%, and GTA-15%—acidophilus milk with 5, 10, and 15% of green tea infusion, respectively (bars denote standard error of the mean)
tea supplementation) of all starter microbiota. As revealed by the analysis of variance
(data not shown), the green tea additive did not affect the count of streptococci in
bioyogurts. Some fluctuations observed in their number in the plain bioyogurt (NY)
and the one containing 5% of green tea infusion (GTY-5%; Fig. 3a) during storage were
insignificant. In bioyogurts with higher levels of tea incorporation, the concentration of
cocci was stable within the studied period.
Statistical analysis revealed that the number of bifidobacteria was unaffected by the
bioyogurt type, but it is worth to emphasize that the count of these probiotic bacteria
remained at a higher level for a longer period of time in fermented milks enriched with
green tea (decrease below 7 log cfu g−1 after 3 weeks in bioyogurts with green tea vs.
1 week for NY). The concentration of B. animalis subsp. lactis BB-12 in overall
population of starter microorganisms determined in the obtained probiotic yogurts
ranged between 1 and 5% for nonsupplemented and fortified with 5 and 10% of GTI
bioyogurts, whereas in GTY-15%, the average bifidobacteria share was ∼10%.
According to the manufacturer’s stated colony-forming unit counts, the lactobacilli to
bifidobacteria to streprococci ratio at inoculation should have been 2:2:1. Thus, major
changes occurred in the strain ratio during fermentation.
The effect of GTI incorporation into the bioyogurt formulation on the viability of
L. acidophilus LA-5 was dose-dependent. The higher the dosage of GTI, the lower number
of these bacteria evaluated in fermented milk. The 5% green tea supplementation resulted
in significantly higher number of lactobacilli (by almost one log cycle) than higher doses of
GTI. In relation to the plain bioyogurt, the difference was negligible.
Acidophilus milks contained higher amounts of lactobacilli than bioyogurts (8.72–
9.02 vs. 7.21–8.29 log cfu g−1). In the case of milk fermented with L. acidophilus LA-5
in monoculture, the green tea infusion at 10 and 15% influenced the higher level of
336
D. Najgebauer-Lejko
bacteria than 0 or 5% treatment. The milks which contained 5 and 15% of GTI were
very stable as regards lactobacilli content within the study period, whereas in NA and
GTA-10%, some fluctuations in the number of these bacteria were observed.
3.4 Effect of tea addition on the sensory properties of probiotic milks
The incorporation of tea extracts into the bioyogurt had no significant influence on the
notes received in the sensory evaluation (Table 1). The notes fluctuated in the range of
4.43–4.86 and were slightly lower at the end of the experiment, although the storage
time was an insignificant factor as regards this feature. Acidophilus milks were worse
appreciated by the panelists than bioyogurts (3.52–4.53), which was connected with
less acceptable flavor as well as consistency and general appearance mainly due to
visible whey separation (data not shown). In this case, fermented milks supplemented
with tea received lower notes than plain acidophilus milk and with one exception
(GTA-15% at the 21st day), the higher tea additive was related to lower notes.
4 Discussion
The main phenolic compounds present in green tea responsible for its high antioxidant
potential are catechins: (−)-epigallocatechin gallate (EGCG), (−)-epigallocatechin
(EGC), (−)-epicatechin gallate (ECG), and (−)-epicatechin (EC) as well as gallic acid
(Cabrera et al. 2003). Research data usually focus on EGCG as the very powerful tea
antioxidant. EGCG was the most abundant flavan-3-ol compound detected in green tea
infusion in the present study. Milk also possesses some antioxidant activity resulting
from the presence of such components as bioactive peptides derived from both caseins
and whey proteins, lactoferrin, urate, ascorbate, α-tocopherol, β-carotene, coenzyme
Q10, and enzymatic systems (superoxide dismutase, catalase, and glutathione peroxidase), which is a property that in fermented milk can be further improved as starter
microorganisms also possess some antioxidant potential (Chen et al. 2003; Kullisaar
et al. 2002). Much more higher DPPH radical-scavenging and ferric-reducing abilities
of green tea resulted in certain enhancement of this property in fermented milks with
every increase of tea supplementation.
The results obtained for acidity measurement in this study, which suggest that GTI
slowed down the acidity development during fermentation and storage, are opposite to
those obtained for the conventional yogurt, where 5–15% green tea additive (regardless
of the concentration) resulted in significantly lower pH values and simultaneously
higher L. delbrueckii subsp. bulgaricus count when compared with the plain yogurt
(Najgebauer-Lejko et al. 2011). On the other hand, Jaziri et al. (2009) demonstrated that
green tea extract had no effect on the lactic acid concentration and bacteria survival in
yogurts, while data from Gaudreau et al. (2013) are at least partially in agreement with
the detrimental effect of higher concentrations of tea extracts on the growth of
lactobacilli in bioyogurts as observed in the present study. In ABT yogurt culture used
in the present study, L. bulgaricus is excluded, which significantly reduces the pH
decrease during production and storage as this species is a better lactic acid producer
than L. acidophilus and Bifidobacterium sp. (Lourens-Hattingh and Viljoen 2001;
Shihata and Shah 2002).
Probiotic milks with green tea
337
To exert the beneficial health effects, the amount of probiotic bacteria in the food
product should be adequately high, i.e., 106–108 cfu mL−1 throughout the entire shelf
life (Ghoddusi and Hassan 2011). The level of L. acidophilus and B. animalis ssp. lactis
evaluated during the whole storage period (21 days) in this study met this criterion.
Enhanced viability of bifidobacteria in tea-supplemented yogurts during the first
2 weeks of refrigerated storage may be connected with higher pH values of bioyogurts
containing higher amounts (10 and 15%) of green tea (pH≥4.6) as growth of most
strains of bifidobacteria is retarded at pH values below 4.6 (Lourens-Hattingh and
Viljoen 2001).
In the study conducted on milk fermented with traditional yogurt culture, the
positive effect of green tea infusion (the same tea and preparation procedure as in the
present study) at the concentration of 10 and 15% was observed, whereas 5% supplementation negatively affected the population of streptococci when compared with
natural yogurt (Najgebauer-Lejko et al. 2011). On the contrary, Jaziri et al. (2009)
reported that green or black tea had no effect on the lactic acid bacteria in yogurt. This
suggests that the effect of tea supplementation on the growth and survival of selected
starter microorganisms in milk systems, among other factors (type of tea and its
composition, procedure of tea yogurt preparation, etc.), depends also on the composition of starter microbiota used for fermentation.
In the present study, LA-5 probiotic strain of L. acidophilus was used for milk
fermentation in the monoculture and in the coculture with S. thermophilus and B. lactis
BB-12. The viability of lactobacilli was influenced by both the green tea concentration
and type of fermented milk product. These findings are in concordance with the
observations of Tabasco et al. (2011) that the sensitivity of lactic acid bacteria (LAB)
and bifidobacteria to the phenolic compounds depends on the bacterial species and
strain as well as chemical structure and concentration of the polyphenols. In their study,
the growth of tested L. acidophilus LA-5 strain was negatively affected by the addition
of flavan-3-ol-enriched grape seed extract. On the contrary, Hervert-Hernández et al.
(2009) reported the stimulatory effect of grape phenolic extract and some of its pure
components (tannic acid, catechins) on the growth of probiotic lactobacilli
(L. acidophilus CET 903). Almajano et al. (2008) showed the inhibitory effect of
extracts of different teas against food-borne pathogens, e.g., Bacillus cereus,
Micrococcus luteus, and Pseudomonas aeruginosa, whereas L. acidophilus exhibited
exceptional resistance to all extracts studied. The different response of L. acidophilus in
its pure culture and coculture to the different concentrations of green tea extract applied
needs to be considered taking into account the interactions between starter microorganisms in ABT yogurt. The effect of the accompanying species used to ferment milk
on the survival of L. acidophilus and Bifidobacterium spp. was reported by LourensHattingh and Viljoen (2001). Moreover, as some species of the lactobacilli were
demonstrated to have the ability to metabolize phenolic compounds (Lee et al. 2006;
Rodríguez et al. 2009), the effect of the potential phenolic metabolites cannot be
excluded.
Saxelin and coauthors (2003) stated that despite the health benefits, fermentation of
milk with pure probiotic strain may result in a product with texture and taste that does
not meet the consumer approval; therefore, the common practice is to use probiotic
strains together with standard starter cultures, e.g., yogurt. This often takes place in the
case of acidophilus milk as L. acidophilus is a homofermentative bacterium that gives
338
D. Najgebauer-Lejko
plain acid flavor, which is frequently perceived as too sour with lack of real aromatic
flavor (Lengkey and Adriani 2009). The results of our study suggest that the main taste
sensations found in green tea, such as astringency and bitterness (Chaturvedula and
Prakash 2011), did not compose well with the taste of acidophilus milk. The possible
phenolic metabolites (not studied herein) may also influence sensory characteristic of
the product.
5 Conclusion
The incorporation of GTI in a dose-dependent manner increased antiradical and ferricreducing power of probiotic milks with no significant differences between the
bioyogurts and acidophilus milks with the same level of supplementation. Bioyogurts
with different levels of green tea infusion did not vary as regards the average counts of
S. thermophilus and B. animalis ssp. lactis BB-12, but GTI at all applied concentrations
maintained the viability of bifidobacteria at the level above 7 log cfu g−1 for an
additional 2 weeks compared to the plain bioyogurt. Green tea at the concentration of
5% was more beneficial for the viability of L. acidophilus in bioyogurt, whereas 10 and
15% positively affected LA-5 growth in monoculture.
In summary, the results of the present study suggest that green tea can be successfully employed as a functional supplement for probiotic milks, adding extra value to the
known health benefits of probiotics, but the proper amount of tea additive and cultures
for milk fermentation need to be carefully chosen.
Acknowledgments The author would like to thank Robert Socha for HPLC study. The present research was
supported by the funding of Polish Ministry of Science and Higher Education (DS/3700/WTŻ/13).
Open Access This article is distributed under the terms of the Creative Commons Attribution License which
permits any use, distribution, and reproduction in any medium, provided the original author(s) and the source
are credited.
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