Raspberry and Strawberry Addition Improves Probiotic Viability in Yogurt and Possess Antioxidant Activity

This study aimed to i) investigate probiotic potentials of raspberry and strawberry addition in yogurts, ii) explore antioxidant activity of berries extracted by microwave using oxygen radical absorbance (ORAC), and 2,2-diphenyl-1-picryhydrazyl radical (DPPH) as well as iii) determine the total phenolic content (TPC) of the berries. The probiotic potentials of those berry additions into yogurts containing different probiotics were determined by subsequent viable microorganism counts in each yogurt trial using selective media, pH and total titratable acidity (TTA) during 28 days of cold storage at 4oC. Viable microbial counts in yogurt trials containing probiotic Lactobacillus acidophilus and raspberry increased (P<0.05) for 21 consecutive days of cold storage. The pH levels decreased (P<0.05) as the TTA increased over 28 days of cold storage in all yogurts containing the berries. ORAC results showed that raspberry had higher antioxidant activity (505.72 μmol TE/100g of fruit) than strawberry (495 μmol TE/100g of fruit). Also, DPPH scavenging activity results showed that raspberry (86.11%) had higher antioxidant activity than strawberry (85.69%). There was not a significant (P<0.05) difference in TPC values of raspberry (0.20 g GAE/kg) and strawberry (0.18 g GAE/kg). This study suggests that both berries have potential as a source of prebiotics with antioxidant activity for future functional foods and nutraceutical applications.


Introduction
Berries like strawberry (Fragaria × ananassa Duch.) and raspberry (Rubus idaeus L.) are traditionally known as part of Nordic diet (Willett et al., 1995).They are an important source of fiber and bioactive compounds like polyphenolics and well recognized because of their positive health effects on human health, especially in the prevention of various oxidative stress associated diseases like cancer (Del Rio et al., 2013).
Prebiotics are nondigestible food carbohydrates such as fructooligosaccharides (FOS) and inulin that improve host health by stimulating the growth and activity of bacteria present in the colon (Gibson & Roberfroid, 1995).Those bacteria are known as probiotics (Kailasapathy & Chin, 2000).The most common probiotics are lactic acid bacteria (LAB) and bifidobacteria (Huebner, Wehling, & Hutkins, 2007;Khurana & Kanawjia, 2007;Ötles, Çagındı, & Akçiçek, 2003).The combination of prebiotics and probiotics which is called synbiotics can be used to manage the microflora in the gut, and enhance survival of probiotics by stimulating growth/activity of bacteria in the colon by prebiotics (Gibson & Roberfroid, 1995;Khurana & Kanawjia, 2007).Examples of synbiotics are bifidobacteria (probiotics) and FOS (prebiotics) as well as lactobacilli and lactitol (Collins & Gibson, 1999).Both prebiotics and probiotics are mostly used in fermented dairy products like functional food product worldwide.Therefore, more research is needed to develop new high value bio products and increase their potential use in functional foods or nutaceuticals (Figueroa-Gonzzalez, Quijano, Ramirez, & Cruz-Guerrero, 2010).
The objectives of this study were to i) evaluate probiotic potentials of raspberry and strawberry addition in yogurts, ii) investigate antioxidant activity of microwave extracts of the both fruits using ORAC, DPPH, and iii) determine the TPC values of berries.The probiotic potentials of those fruit addition into yogurts containing decanted and 5 mL of sterile water was added.A hemacytometer was used to count the bacteria until a concentration of about 6.5 log cfu/mL was reached.

Yogurt Preparations
Homogenized (3.25%) milk (commercial source in Ottawa, ON) was heated until the temperature reached 85 C for 15 minutes.Then, the pasteurized milk was cooled in a water bath and kept at 42 C (Espírito Santo et al., 2010).Twelve different yogurt treatments were prepared; 4 with raspberry, 4 with strawberry and 4 without berry (control) as shown in Table 1.For each test tube, 50 mL of pasteurized milk and 1 mL of starter culture (microorganism diluted with milk) was added.The probiotics (1 mL) were added to the respective yogurt treatments as presented in Table 1.The treatments were incubated at 42 C until yogurt was formed, once formed the tubes were stored at 4 C in the fridge.All analyses were made in triplicate.

Microbiological Analyses
Viable bacteria counts were performed on day 1, 7, 14, 21 and 28 in triplicate by following the study of Espírito Santo et al. (2010).Serial dilutions (10 -1 to 10 -5 ) were made for each yogurt treatment using a 1: 9 ratio.For each dilution, 10 L was plated onto agar dishes using the spread plate method.
The starter cultures and probiotics were enumerated on selective media according to the method by Vinderola et al. (1999).L. delbrueckiii subsp bulgaricus was enumerated on MRS (pH 5.4) agar and grown under aerobic conditions for 72 hours at 42C (Gonçalves, Freitas, Nero, & Carvalho, 2009;Vinderola & Reinheimer, 1999).S. thermophilus was enumerated on M17 agar and grown under aerobic conditions for 24 hours at 37 C (Vinderola & Reinheimer, 1999).L. acidophilus was enumerated on T-MRS agar and grown under aerobic conditions for 48 hours at 37 C (Vinderola & Reinheimer, 1999).B. lactis was enumerated on LP-MRS agar and grown under anaerobic conditions in BBL GasPak™ System (GasPak System-Oxoid, Basingstoke, UK) for 48 hours at 37 C (Vinderola & Reinheimer, 1999).The number of colonies was counted and the number of cells was converted into log cfu (colony forming units) per mL.

pH and TTA
Both pH and TTA values of each yogurt treatment (Table 1) was measured on day 1, 7, 14, 21 and 28 according to the method by Espírito Santo et al. (2010).The pH was measured using the Denver Instrument UB-5 pH meter.TTA value was determined by mixing of 1 mL of yogurt with 9 mL of sterile water (1: 9) which was titrated with 0.1 M NaOH and 0.1% phenolphthalein colour indicator (Behrad, Yusof, Goh, & Baba, 2009).The amount of acid produced during fermentation was expressed as TTA%.All analyses were made in triplicate.

Microwave Extraction of Water Extractable Material (WEM)
Raspberries and strawberries were crushed using a juice processor and stored in Ziploc bags in the freezer at -20 C prior to analysis.WEM was extracted by using microwave.The samples (5 g of each berry samples) and 50 mL of distilled water were added into a quartz vessel and placed into the CEM STAR System 2 microwave digestion system (CEM Corporation, Matthews, NC, USA) at 90 C for 30 minutes.The mixture was then cooled and centrifuged at 4000 rpm for 20 minutes at room temperature (23 C).The supernatant was collected and stored at -20 C in the freezer until further analysis (Liazid, Palma, Brigui, & Barroso, 2007).All analyses were made in triplicate.

ORAC
The antioxidant activity of raspberry and strawberry WEM was measured using ORAC assay by following the procedures of Huang et al. (2002) and Hosseinian et al. (2007).The FLx800 TM Multi-Detection Microplate Reader with Gen5 TM software by BioTek Instruments was used to carry out the assay.Basically it consists of using 2,2'-Azobis (2-amidinopropane) dihydrochloride (AAPH) as the free radical generator, Trolox (water-soluble α-tocopheral (Vitamin E) analogue) as the standard and fluorescein working solution as the fluorescent probe (Huang, Ou, Hampsch-Woodill, Flanagan, & Prior, 2002;Wang & Lin, 2000).Five different concentrations of Trolox standard (6.25, 12.5, 25, 50 and 100 M) and two concentrations (200 and 100 M) of rutin control were prepared.For each ORAC run, a 96 micro-well plate was prepared; including 20 µL of buffer (blank), Trolox (standards), sample, and rutin was loaded into designated wells.In each well, 120 µL of fluorescein working solution was added to incubate at 37 C for 20 min.Then, 60 µL of AAPH peroxyl radical generator was added into each well to make a total well volume of 200 L.The fluorometric microplate reader was used at an excitation wavelength and emission wavelength of 485 nm and 528 nm, respectively.The ORAC value was calculated by using the area under the curve (Net AUC) of the sample and the equation of the line from the Trolox standard curve.ORAC values were expressed as micromole Trolox Equivalents/100 g berry.All analyses were carried out in triplicate.

DPPH
The antioxidant activity of raspberry and strawberry WEM was determined using the DPPH scavenging activity assay (Li, Hydamaka, Lowry, & Beta, 2009).Briefly, 200 L of sample was mixed with 3.8 mL of DPPH solution (60 M).The absorbance (A) of the mixture was measured at a wavelength of 515 nm at 0, 5, 10, 15, 20, 30, 40, 50, 60 minutes using the UV-Visible SpectraMax Plus384 spectrophotometer in triplicate.Absorbance was measured against a blank of methanol.The antioxidant activity was calculated as percent discoloration as shown in the following equation.All analyses were made in triplicate.

Extraction of Phenolics and TPC Analysis
Extraction: Phenolic compounds from raspberry and strawberry were extracted according to the method of Li et al. (2009).Each berry sample (1.0 g) was mixed with 15 mL of ethanol (95%)/1N HCl (85:15, v/v) solution and the mixture was stirred for 6 hours at room temperature (23C).Then the mixture was centrifuged at 4,000 rpm for 15 minutes at 5C.The supernatant was collected and stored at -20C in the freezer under further analysis.
TPC analysis: 200 L of each berry extract was mixed with 1.9 mL of 10 fold diluted FC reagent.After 5 minutes at room temperature (23C), 1.9 mL of a 60 g/L sodium carbonate solution was added.The absorbance was measured after 120 minutes of incubation at room temperature (23C) at 725 nm against a blank of distilled water using the UV-Visible SpectraMax Plus384 spectrophotometer.The absorbance was measured in triplicate and the results were expressed as gallic acid equivalents per gram of sample.

Statistical Analyses
All experiments were conducted in triplicates by means of Analysis of variance (ANOVA) with Statistical Analysis System (SAS, version 9.2, SAS Institute Inc., Cary, NC).Duncan's Multiple Range test was used when significant (P<0.05)mean comparison was performed.

Berry Concentrations
The best raspberry and strawberry concentration that could be added into milk was determined by adding different amount of berry to milk and maintain a stable yogurt product that would be acceptable to the consumers as palatable yogurt (Espírito Santo et al., 2010).Therefore, 3% berry addition in yogurt was determined as the best concentration to be employed for the all yogurt trials (Table 1) since there was no sign of syneresis (separation of water from gel) (Figure 1A and B).

Viable Microorganism Counts With Selective Media
The viable microorganism counts of Streptococcus thermophilus and Lactobacillus delbruekii subsp.bulgaricus in all yogurt trials (Table 1) are shown in Table 2 as log cfu mL -1 .Both counts showed no significant (P<0.05)difference among treatments (Table 2) after 1 day of cold storage and varied from 7.18 to 7.82 log cfu mL -1 .). e an electron-transport chain which means that oxygen is not going to be reduced properly and exposure to oxygen can lead to cell death due to metabolite accumulation (Talwalkar & Kailasapathy, 2004).The growth of bifidobacteria can be affected by other microorganisms that are present within the yogurt which can possible restrict its growth (Kailasapathy & Chin, 2000).The decrease could also be due to a reduction in sugars present in the yogurt which in turn provides fewer nutrients for the bacteria to consume (Agil & Hosseinian, 2012).
On day 7, the pH among all yogurt treatments ranged from 6.06 to 6.50 (Table 3).There was not a significant difference (P<0.05) in pH among treatments containing berry compared to the control.The TTA% among all yogurt treatments ranged from 0.16 to 0.25 (Table 4).Yogurt treatments containing raspberry (1R-4R) were significantly higher (P<0.05)than the control.Yogurt containing strawberry with L. acidophilus (2S) and strawberry with both probiotics (4S) were significantly higher (P<0.05)than the control.
On day 14, the pH among all yogurts treatments ranged from 5.77 to 6.47 (Table 3).Yogurt containing raspberry (1R) had a significantly lower (P<0.05)pH than the control.The TTA% among all yogurt treatments ranged from 0.21 to 0.49 (Table 4).There was a significant higher (P<0.05)lactic acid content in yogurt containing raspberry with the exception of yogurt with raspberry and both probiotics (4R) compared to the control.On day 21, the pH among all yogurt treatments ranged from 5.41 to 6.48 (Table 3).Yogurt that contained raspberry (1R), raspberry and B. lactis (3R), raspberry and both probiotics (4R) and strawberry and both probiotics (4S) had a significantly lower (P<0.05)pH compared to the control.The TTA% among all yogurt treatments ranged from 0.40 to 0.60 (Table 4).In all yogurts containing raspberry (1R-4R) and strawberry (1S-4S), the lactic acid content was significantly higher (P<0.05)than the control.
On day 28, the pH among all yogurt treatments ranged from 5.10 to 6.04 (Table 3).Yogurt containing raspberry (1R-4R) and strawberry (1S-4S) had a significantly lower (P<0.05)pH compared to the control.The TTA% among all yogurt treatments ranged from 0.36 to 0.60 (Table 4).All yogurt treatments containing berry had a significantly higher (P<0.05)lactic acid content compared to the control with the exception of yogurt containing raspberry (1R).
After 28 days of cold storage the pH in yogurts containing raspberry and strawberry were lower than control yogurts (P<0.05).The TTA% in yogurts with raspberry and strawberry were higher than control yogurts which has no berry addition.Yogurts that contain berry possibly have a greater decrease in pH due to microorganisms that are more active in the presence of berries (Kailasapathy et al., 2000).The increase in TTA indicates that during the growth of bacteria there is lactic acid production (Agil & Hosseinian, 2012).

ORAC
Raspberry had higher antioxidant activity (P<0.05)compared to strawberry with ORAC values of 505.72 µmol TE/100 g of fruit and 495 µmol TE/100g of fruit respectively (Table 5).In the study of Wang and Lin (2000), the antioxidant activity of fruits and leaves of different genotypes and development stages of blackberry, raspberry and strawberry fruits and was investigated.The ORAC values of fresh red and black raspberries from different cultivars ranged from 7.8 to 33.7 mol of TE/g during different stages of maturity (Wang & Lin, 2000).The ORAC values of fresh strawberries from different cultivars ranged from 12.2 to 17.4 mol of TE/g during different stages of maturity (Wang & Lin, 2000).The antioxidant capacity was lower in strawberry compared to blackberries and raspberries (Wang & Lin, 2000).The higher the phenolic and flavonoid content, the higher the antioxidant activity of the fruit (Liu et al., 2002). www.ccsen

Table 1 .
The experimental design used to evaluate the effects of raspberry and strawberry addition on probiotic viability in different yogurt trials

Table 5
strawberry are preliminary and further study is required to investigate how polysaccharides and other bioactive compounds extracted from raspberry and strawberry will affect the growth of starter and individual pure probiotic bacteria.