Effects of Tea (Camellia sinensis) Phytochemicals on the Yoghurt Cultures (Lactobacillus bulgaricus and Streptococcus thermophilus) During Development and Storage of Tea Fortified Yoghurts

Tea phytochemicals have been reported to exhibit potent antimicrobial activity. This current study reports the ability of Streptococcus thermophilus and Lactobacillus bulgaricus to grow, survive and multiply in the presence of tea phytochemicals during development and storage of tea fortified yoghurts. Two Kenyan tea varieties clone TRFK 6/8 (standard black quality tea) and clone TRFK 306/1 (newly developed purple leafed tea clone), were processed as aerated and non-aerated teas and used to develop tea fortified yoghurt. The teas were added at concentrations of 0, 1, 2 and 4 g in 250 mL volumes of milk (w/v) which was predetermined by sensory tests. The results showed that tea phytochemicals at the added ranges had no adverse effect on the growth of Lactobacillus bulgaricus and Streptococcus thermophilus; however, yoghurt setting time was prolonged at a mean time of 4.11, 5.22, 7.29 and 8.26 hrs respectively for tea concentrations of 0, 1, 2 and 4 g/250 mL milk. The mean microbial load for Lactobacillus bulgaricus and Streptococcus thermophilus in black, green and purple tea yoghurts were also inversely proportional to the concentration of the added teas. In the developmental stages of tea fortified yoghurts Lactobacillus bulgaricus range was 0.52-1.58 × 10 CFU/mL while Streptococcus thermophilus range was 2.53-3.51 × 10 CFU/mL, during storage the growth patterns were different between the cultures. The recorded mean values range for Lactobacillus bulgaricus was 2.79-4.35 × 10 CFU/mL while Streptococcus thermophilus mean range was 2.57-3.47 × 10 CFU/mL Phytochemicals traced in the product had concentration values below 5 × 10 μgmL. In conclusion, it was possible to develop probiotic tea fortified yoghurt containing tea phytochemicals with unlimited health benefits using different the different tea clones.


Introduction
Kenya is a tea growing country and produces black tea as its major trading product commodity.In addition only 5% of this is consumed within the country while the remaining 95% is exported (Ochanda, 2010).In recent years, black tea prices have been on the decline while other speciality teas and tea products have been on the rise.In order to address this issue and turn around the tea industry product diversification and value addition is required as is done in developed countries (Ochanda, 2010;Lelgo, Kamunya, Ochanda, & Wanyoko, 2011;Trienekens, 2011).Tea is usually used to fortify various foods due to its low pH (4.2) (Yilmaz, 2006), high polyphenol, antioxidant and potent antimicrobial activity (Almajano, Carbó, Jiménez, & Gordon, 2008;Jaziri, Slama, Mhadhbi, Gannoun, & Othman, 2005;Koech et al., 2013;Oh, Jo, Cho, Kim, & Han, 2013).This research focused on value addition of tea through the development of tea fortified yoghurts.The tea fortified yoghurts was intended to have double health benefits including the yoghurt starter cultures' probiotic properties and tea phytochemicals both of which are associated with unlimited health benefits.However there was a possible challenge, in the development and production of the tea fortified yoghurt with regard to the growth and survival of the yoghurt cultures owing to teas inherent antimicrobial activity.

Yoghurt
Yoghurt is a dairy product obtained by lactic acid fermentation of milk by Lactobacillus delbrueckii ssp.Bulgaricus and Streptococcus thermophilus which acidify milk and synthesize aromatic compounds (Jaziri et al., 2005;Serra, Trujillo, Guamis, & Ferragut, 2009).It contains viable micro flora of starter origin at the time of consumption, with minimum values between 10 6 and 10 8 CFU/mL (Serra et al., 2009;Lourens-Hattingh & Viljoen, 2001).The lactose in the milk is converted to lactic acid when fermented.Lactic acid acts on the protein in the milk to give yoghurt its texture and characteristic tang (Rao, Tanksale, Ghatge, & Deshpande, 1998).The milk is usually heat treated to pasteurization temperatures and time to kill all pathogenic and some spoilage microorganisms and to change the physical structure of the milk proteins so that they set together instead of forming curds.Afterwards it is cooled to 45 ºC, the starter culture bacteria are then added and the milk is incubated at that temperature for approximately 4-4.5 hr to ferment.The product exists in a variety of forms i.e. stirred, set, frozen and liquid yoghurt.Good packaging is essential for preservation of the product (Marth & Steele, 2001).Yoghurt is rich in the macronutrient protein, and other micronutrients namely calcium, riboflavin, vitamin B6 and vitamin B12 (Campbell, 2003).Moderately lactose-intolerant people can consume yoghurt without ill effects.It prevents antibiotic-associated diarrhea, promotes good gum health and low-fat yoghurt promotes weight loss (Jaziri et al., 2005).Nutraceuticals such as high fiber, low fat yoghurt also exist (Jaziri, Slama, Mhadhbi, Urdaci, & Hamdi, 2009).Food consumers the world over are showing increasing interest in such health-promoting foods in the diet (Varga, 2006).

Antimicrobial Activity of Tea Extracts
The antimicrobial activity of tea against some bacteria and fungi has been demonstrated by several researchers (Arakawa, Maeda, Okubo, & Shimamura, 2004;Goto et al., 1998;Hamilton-Miller & Shah, 1999;Hamilton-Miller, 1997, 2001;Ikigai, Nakae, Hara, & Shimamura, 1993;Koech & Wachira, 2013;Navarro-Martínez et al., 2005;Stapleton, Shah, Anderson, et al., 2004;Stapleton, Shah, Hamilton-Miller, et al., 2004;Zhang & Rock, 2004).Depending on the type of processing, different tea have different active compounds responsible for these microbial activities.The major active compound for these direct antimicrobial effects in non-aerated green tea and its extracts appears to be the tea phytochemicals Epigallocatechin gallate (EGCG).In black tea and black tea extracts, the active biomolecule which is thought to be responsible for the antibacterial activity is the catechin dimer theaflavin (TF) and its gallate while in purple tea it is the anthocyanins in general (Cisowska, Wojnicz, & Hendrich, 2011).The commercially available green tea extracts have been formulated and incorporated as active ingredients in various products such as antiseptic creams, mouthwashes, hand washes, facemasks among other products in order to prevent infections (Ochanda, 2010).Tea extracts have also been introduced into vacuum cleaner filters in order to reduce airborne contamination with microorganisms (Yukihiko Hara, 2001).Products having catechin-mediated photo-protection effects of human skin against bacterial infection has also been claimed (Hsu, 2005) and some researchers have found that topical tea ointment was as effective for the treatment of impetigo as a topical antibiotic mixture (framycetin and gramicidin) or an oral cephalexin (Sharquie, Al-Turfi, & Al-Salloum, 2000).
Due to the antimicrobial activity associated with tea components, an assay on the effect of incorporating tea in yoghurt was investigated, by looking into the interaction of tea phytochemicals with yoghurt starter culture during the development and storage of the tea fortified yoghurts.

Methods
The experiment was a 3 × 4 factorial embedded on a Randomized Complete Block Design (RCBD) consisting of yoghurts fortified with black, green and purple tea at 4 varying concentrations of 0, 1, 2, and 4 g/250 mL (w/v).The control yoghurt had all the ingredients used to prepare the yoghurts except the teas

Materials for Yoghurt Processing
The ingredients included; Teas (aerated and non-aerated teas from two Kenyan tea varieties Clone TRFK 6/8, Purple tea clone TRFK 306/1) obtained from Tea Research Institute, sugar, whole milk, yoghurt starter culture (Mixture of L. bulgaricus and S. thermophilus), skimmed milk powder, packaging material (plastic bottles).

Development and Storage of Yoghurts
Fresh milk was homogenized, fortified with the teas at rates of 1 g, 2 g and 4 g/250 mL (w/v) and then pasteurized to produce black, green and purple tea fortified yoghurts.Each set of yoghurt had a control without tea.The milk was infused in the teas then pasteurized by boiling at 90 ºC for 3 min, filtered, cooled to 45 ºC and inoculated with 6.5% (w/v) lactic starter culture at a ratio of 2:1 Streptococcus salivarius ssp.thermophilus and L. delbrueckii ssp.bulgaricus.Incubation was done for 4-8 hrs at 45 ºC (optimum temperature) which brought the pH of the yoghurts to between 4.5-4.6.Yoghurts were cooled and stored at 4 ºC.The viscosity and pH of the yoghurts were monitored from inoculation to the setting stage of the products.Enumeration of starter culture microorganisms of yoghurts during post-acidification period was also carried out weekly for 63 days to determine the interaction between tea and yoghurt cultures.

Determination and Measurement of Yoghurt Setting Time
The setting of the tea fortified yoghurts and their controls was determined by pH Meter assays during development at values between pH 4.5-4.6 which was determined according to AOAC method (AOAC, 2012).

Enumeration of Yoghurts Microbial Starter Culture
Aseptic sampling was done and samples diluted 9 times.One (1) mL of diluted sample was transferred into 9 mL of sterile peptone water.Further dilutions were appropriately made as required.The standard spread plate method was used to determine the viable cell counts of starter organisms (AOAC, 2012).M17 agar at pH 7.1 ± 0.1 was used to culture S. thermophilus, incubated at 45 ºC for 48hr under aerobic conditions before enumeration.S. thermophilus formed lenticular colonies.Colony forming units (CFU) enumerated were expressed per milliliter (mL) of yoghurt sample, and used to report survival of streptococci.Acidified MRS agar at pH 6.5 was used for the culture of L. bulgaricus, incubated at 45 ºC for 48hr under aerobic conditions.L. bulgaricus formed 1-3 mm diameter colonies.The counts were expressed as colony forming units (CFU) per mL of sample.

Statistical Analysis
Analysis of variance (ANOVA) and mean comparisons of yoghurt setting time and of surviving starter cultures of S. thermophilus and L. bulgaricus during product development and storage was determined at P ≤ 0.05 with the system programme SAS (Arthur, 2013).

Results
Tea fortified yoghurt cultures, Lactobacillus delbrueckii ssp.bulgaricus and Streptococcus salivarius ssp.thermophilus are displayed in Figure 1.The cultures survived the added tea phytochemicals from the black, green and purple teas during yoghurt development.

Phytochemical Determination in Tea Fortified Yoghurts
The developed products of tea fortified yoghurts containing black, green and purple teas at varying tea concentrations were assayed for polyphenol compounds and the results were positive.Black tea fortified yoghurts contained at least an epigallocatechingallate (EGCG) pigmentation while green tea fortified yoghurts contained both Epicatechin (EC) and epigallocatechin (EGC) pigments.Purple tea on the other had contained Epicatechin (EC) and some unidentified pigments which could have been anthocyanins (Figure 4).However the concentration range was below 5 × 10 2 µgmL -1 .After inoculation the growth of the yoghurt in the teas were as follows; Lactobacillus bulgaricus in black tea fortified yoghurts, 1.53, 1.19, 0.67 and 0.57 × 10 7 CFU/mL; green tea fortified yoghurt 1.58, 0.99, 0.70 and 0.52 × 10 7 CFU/mL; purple tea 1.58, 1.26, 0.72 and 0.60 × 10 7 CFU/mL respectively for 0, 1, 2 and 4 g/250 mL concentration in the first 10hr of yoghurt development.Lactobacillus bulgaricus in black tea had a range of 0.11 -0.12 × 10 7 CFU/mL for 4g and 0g for time 0hr to 1.11 -2.86 10 7 CFU/mL for 4 g and 0 g respectively for time 10hr with a mean of 1.90 × 10 7 CFU/mL showing a suppression effect to the yoghurt culture with increasing addition of tea.. Green tea range was from 0.10-0.12for 4g and 0 g/250 mL tea concentration at time 0hr to 1.04-2.96× 10 7 CFU/mL for 4g and 0 g/250 mL with a mean of 1.54 × 10 7 CFU/mL Purple tea range was from 0.11-0.12× 10 7 CFU/mL for 4 g and 0 g/5 mL at time 0 hr to 1.15 and 2.96 × 10 7 CFU/mL for 4g and 0 g/250 mL tea concentration with a mean value of 1.98.All the results show indicate a reduction in the starter culture proliferation rate with each addition of tea.However teas did not hinder the growth of starter culture (Figure 2ai-ci).
Streptococcus thermophilus growth patterns in the presence of tea also exhibited suppression with each addition of tea.Black tea fortified yoghurt range was 1.03-1.04× 10 9 CFU/mL for 0-4 g/250 mL for time 0hr to values of 4.92-5.55× 10 9 CFU/mL 4-0 g/250 concentration of tea.Green tea range was 1.02-1.03for 0g and 4 g/250 mL at time 0hr to 5.03-5.47× 10 9 CFU/mL at time 10 hr for 4 g and 0 g/250 mzL respectively.Purple tea had an initial mean of 1.03 × 10 9 CFU/mL for 4 g and 0 g/250 mL concentration to 5.07-5.47× 10 9 CFU/mL for 4 g and 0 g/25 mL respectively.However the acceptable level (Sera et al., 2009) for the starter culture load was met at the end of the development period of 10 hr (Figure 2aii-cii).

Starter Culture at Storage
The critical quantities of the cultures that is 10 6 -10 8 CFU/mL for Lactobacillus bulgaricus and Streptococcus thermophilus was achieved and surpassed by 10 1 and 10 2 CFU/mL respectively (Figure 2 a-c and 3a-c) from production date during storage and at the end of the storage period of 63 days.Usually yoghurt has a shelf life of 45 days.
During yoghurt storage the growth patterns were different with Lactobacillus bulgaricus on a low at day 0 to a maximum at day 19 to a decline to a low rate at day 45 and a final lowest peak at day 63.The recorded proliferation rates for the different tea fortified yoghurts were as follows: Black tea fortified yoghurts 3.86, 3.47, 3.25 and 2.79 ×10 7 CFU/mL Green tea fortified yoghurts 4.35, 3.77, 3.28 and 3.07 × 10 7 CFU/mL and purple tea fortified yoghurts, 4.06, 3.62, 2.99 and 2.80 × 10 7 CFU/mL for 0, 1, 2 and 4 g/250 mL milk concentration. .However the counts remained within the accepted range of 10 6 and 10 8 CFU/mL (Serra, Trujillo, Guamis, & Ferragut, 2009).The shelf life for yoghurts is usually set at 45 days which as seen from the graphs is a time period at which optimum counts of the Lactobacillus bulgaricus can be obtained.Black, green and purple tea fortified yoghurts displayed a similar trend.There were significant differences in the microbial counts of controls and that of all the added teas from concentrations 2 and 4 g/250 mL (P ≤ 0.05).On the other had no significant differences were recorded between tea fortification at 0 g/250 mL and 1 g/250 mL tea concentration in all the tea fortified yoghurt.Although the differences in the microbial load of Lactobacillus bulgaricus in the various tea fortified yoghurts and different concentrations was significant (P ≤ 0.05), the counts were still within the accepted range of 10 6 and 10 8 CFU/mL (Figure 3ai-ci) as recommended for yoghurts (Serra, Trujillo, Guamis, & Ferragut, 2009) Streptococcus thermophilus growth pattern was slightly different starting from a maximum at day 0 to a slight decline at day 19 to a steep decline to day 45 with slight stagnation to a final low decline to day 63.Means of proliferation rates were as follow: black tea fortified yoghurts 3.37, 3.18, 2.92 and 2.67 × 10 9 CFU/mL; green tea fortified yoghurts 3.47, 3.11, 2.84 and 2.57 × 10 9 CFU/mL and purple tea fortified yoghurts 3.32, 3.09, 2.96 and 2.76 × 10 9 CFU/mL for 0, 1, 2 and 4 g/250 mL tea concentration.The microbial load for Streptococcus thermophilus was highest in the first 18days and then declined progressively from day 19 to day 45 and then slowly started stagnating with slight decline from day 45 onwards to day 63 of storage.The shelf life of yoghurt at 45 days could also have been informed by this trend observed in the tea fortified yoghurt development since optical culture load are obtained from day 1 to day 45 of storage.However, acceptable microbial load still persisted to day 63 (Figure 3aii-cii), where our studies ended and the microbial load of Streptococcus thermophilus was still within the recommended range of 10 6 and 10 8 CFU/mL (Fadela, Abderrahim, & Ahmed, 2009;Serra et al., 2009).

Phytochemical Determination in Tea Fortified Yoghurts
The assay showed that all the tea fortified yoghurts contained individual phytochemicals that impart health benefits to the teas.The three different tea fortified yoghurts contained different active components.Black tea fortified yoghurts had traces of EGCG, green tea had traces of EGC and EC while purple tea fortified yoghurts had traces of anthocyanin pigments.All these showed that the developed tea fortified yoghurts also possessed teas health benefits as has been found in other tea fortified foods (Jaziri et al., 2005;Yilmaz, 2006).

Conclusion
Tea phytochemicals did not adversely affect the growth and survival of yoghurt starter culture, despite its antimicrobial activity.Setting time was however, prolonged.As the concentration of tea was increased setting time was also increased, with normal setting time for non-fortified yoghurt, being recorded when the pH is between 4.5 and 4.7.Black tea fortified yoghurt had the least setting time while purple fortified yoghurt had the longest setting time at each level of fortification.This time factor needs to be taken into consideration during the processing of these tea fortified yoghurt since it is longer than those of the ordinary yoghurts.
Microbial proliferation and growth pattern was maintained in all the tea fortified yoghurts with respect to the various tea concentrations.Microbial growth was high at low concentration of 0 g/250 mL milk and lowest at concentrations of 4 g/250 mL milk.
Storage stability studies regarding the survival rate of the cultures showed that during the entire storage period, all the tea fortified yoghurts were able to sustain the required starter microbial load at the various levels of tea fortification up to the last experimental day.The microbial load studies lasted 63 days while the average shelf life of yoghurt at retail stores is usually 45 days.The studies revealed that the shelf life of the commercial yoghurt products were set at optimum ranges from day 1 to day 45 although there is still plenty of nutritive value within acceptable ranges of 10 6 -10 8 CFU/mL of microbial load if consumption is continued for 63 days.
The production of the tea fortified yoghurt rich in tea phytochemicals is achievable.These development will increase tea consumption and subsequently increase revenue to tea growers and tea growing regions.