Journal of Food Safety ISSN 1745-4565
GROWTH AND SURVIVAL OF LACTOBACILLUS ACIDOPHILUS AND BIFIDOBACTERIUM BIFIDUM IN PROBIOTIC YOGURTS ENRICHED BY BARBERRY EXTRACT MINA HASSANI1, AKRAM SHARIFI2,4, ALI MOHAMMADI SANI1 and BAHRAM HASSANI3 1
Department of Food Science and Microbiology, Islamic Azad University, Quchan Branch, Quchan, Iran Department of Food Science and Engineering, Faculty of Industrial and Mechanical Engineering, Qazvin Branch, Islamic Azad University, Qazvin, Iran 3 Department of Food Science and Technology, Ferdowsi University of Mashhad, Khorasan, Iran 2
4 Corresponding author. TEL: 1989127335291; FAX: 1982833665279; EMAIL:
[email protected]
Received for Publication August 25, 2015 Accepted for Publication December 21, 2015 doi: 10.1111/jfs.12269
ABSTRACT This study investigated the effect of functional properties of barberry aqueous extract in different concentration of 4 and 5% (w/w) on the growth and survival of probiotic cultures (L. acidophilus, B. bifidum and an equal mixture of the two strains) in the set and stirred yogurt samples. The viable counts of L. acidophilus and B. bifidum in the set and stirred probiotic yogurts containing barberry extract were significantly higher than the control yogurt sample during the storage period. In a single strain of L. acidophilus, the highest viable populations of L. acidophilus in the set and stirred yogurts containing 5% of barberry extract were 1.15 3 108 and 1.1 3 108 on the first day, respectively. Additionally, L. acidophilus counts showed sharp decrease throughout the storage period but its counts held probiotic value of 106 log cfu/gr until Day 21, also the lowest viable count of B. bifidum (9.3 3 105 log cfu/gr) was observed in the control sample at the end of the 21st day in a single strain of B. bifidum.
PRACTICAL APPLICATIONS Fruit and vegetable extracts are widely incorporated in food flavoring and are recently applied as growth-stimulators of probiotic bacteria. The findings of this study indicated that probiotic yogurt samples fortified with barberry extract could be used as an adequate carrier of probiotic bacteria with bacterial counts more than the suggested level.
INTRODUCTION The food industry has witnessed considerable changes in food consumption over the few last decades. Almost all consumers believe that foods can contribute directly to their overall health and wellness (Mollet and Rowland 2005). Nowadays, functional foods are used not only to satisfy hunger and to provide essential nutrients for humans, but also to prevent nutritionrelated diseases and to promote the consumer’s physical and mental well-being (Roberfroid 2000a; Menrad 2003). The increasing demand on such foods can be explained by the high cost of healthcare, the steady increase in life expectancy, and the desire of the elderly people to improve the quality of their future lives (Roberfroid 2000b; Siro et al. 2008).
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In addition to their nutrition role, functional foods provide health promotion benefits to consumers. Furthermore, fruit ingredients which stimulate the growth of probiotic bacteria can increase the success rate of health-promoting probiotic foods. Yogurt is the most popular fermented milk product all over the world. It is characterized by its beneficial effects on human (Chandan 2007). The world production and consumption of yogurt has greatly increased since the 21st century due to the introduction of fruit and vegetableflavored yogurt (Kailasapathy et al. 2008; CHANDAN and KILARA 2010). Fruits and vegetables which are added to yogurt act as prebiotics (fibers) (Allgeyer et al. 2010), as flavoring and coloring agents (Salem et al. 2006; SMITH and 1
GROWTH AND SURVIVAL OF L. ACIDOPHILUS AND B. BIFIDUM
HUI 2008), and as sources of natural antioxidants (Photochemical antioxidants) (Dimitrios 2006). Yogurt contains live probiotic microorganisms, such as L. acidophilus and B. bifidum which reportedly have beneficial health effects. Furthermore, addition of fruit mixes to yogurt can improve its taste and, more importantly, its nutritional value. Recently, the survival of probiotic bacteria during the cold storage have been investigated with regard to a variety of fruit yogurts labeled as probiotic fruit yogurts (Kahkonen et al. 2001). Seedless barberry is produced from hardy shrubs that can grow in regions with poor soil and salt water. With regard to fruit production, Barberries vulgaris L. var. asperma, which is intensively cultivated in various regions of Iran, can be considered as the most well-known variety of barberry (Maskooki et al. 1993). The stem, root bark, and fruit of barberry contain such isoquinoline alkaloids as berberine, which is the main active ingredient of barberry (Gorval and Grishkovets 1999). Berries contain such compounds as gallic acid, flavonoid (Vuorinen et al. 2000; Mullen et al. 2002), anthocyanins, inulin and fructo-oligosaccharides (Alberto et al. 2001) which may support the growth of Lactobacilli and Bifidobacteria. Typical compounds such as sugar and small proteins and other food-specific phytochemicals including phenolics and organic acids present in the aqueous extracts of berries have been also reported to increase the growth of probiotic microorganisms. The growth promoting activity of blueberries and strawberries, as functional food ingredients, on some specific probiotic bacteria strains, namely Lactobacillus rhamnosus, Lactobacillus reuteri and Bacillus lactis has been observed by Sutherland et al. (2009). In fact, among the other compounds of berries which have been reported to increase the growth of certain probiotic bacteria strains include carbohydrates such as pectin and pecticoligosaccharides (Olano-Martin et al. 2002). This study was aimed at studying the growth and viability of probiotic bacteria (L. acidophilous, B. bifidum, and also an equal mixture of the two strains) in set and stirred probiotic yogurts supplemented with different concentration of 4 and 5% (w/w) barberry extracts. The study was further aimed at contributing to the production of new functional foods.
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used for each experiment. The fruit samples were first squeezed by means of a Black & Decker grinder (Model no. JBG60, USA) and then they were placed into a flask with 500 mL of distilled water. The flask was placed in a water bath (Model LAUDA E200, Germany) and stored at 60 6 1C for 2 h. The flask was then separated from the condenser and stored in the dark for 2 h. The barberry extract was filtered in a vacuum Buchner funnel using Whatman Grade 40 filter paper (Maskooki et al. 1993). The filtered extracts were concentrated at 45C using a vacuum rotary evaporator (BUCHI B-480 Water, Flawil, Switzerland). The concentrated extracts were stored in air tight dark glass bottles and kept refrigerated (4C) for further treatments.
Preparation of Yogurt Starter The direct-in vast-set pouches of commercial lyophilized cultures including L. acidophilus La-5 and B. bifidum Bb-12 were bought from Chr. Hansen Company, Denmark. The starter packages were prepared according to the manufacturer’s instructions.
Production of Probiotic Yogurts Set and stirred probiotic yogurts were produced using a standard yogurt manufacturing procedures (Tamime and Robinson 2007). Standardized milk (4% fat and 12% SNF) was homogenized at 60C and then pasteurized at 95C for 15 min. The milk was then cooled at 43C and aseptically inoculated with 0.01% (w/w) of each strain of L. acidophilus and B. bifidum. Additionally, an equal mixture of the two strains with a ratio of 1:1 (0.01% w/w) was added to the milk. Barberry extract in different concentration of 4 and 5% (w/w) was further added to the sterile containers to produce set yogurt and then it was incubated at 37 6 1C for 18 h. Besides, after the preliminary incubation, barberry extracts in concentration of 4 and 5% (w/w) were added to obtain stirred yogurt. All the cultured milk groups were then left for a final fermentation at 43 6 1C. The fermentation was terminated when the acidity level reached a pH value of 4.7. Next, yogurt samples were cooled to 5C and held at this temperature for one day. All the experimental samples were subjected to microbiological analysis during the 21-day of storage period.
MATERIALS AND METHODS
Microbiological Analysis
Seedless barberries (Berberis vulgaris) were harvested from barberry orchards in Ghaen, a town in South Khorasan Province, Iran. Immediately after the samples were cleaned and leaves and stems were removed, they were stored in a cold storage room at 4 6 1C for carrying out the experiments.
The studied samples (25 g) were weighed aseptically in sterile stomacher bags which were diluted with 225 mL of buffered peptone water. Therefore, the first dilution of 1021 was obtained, and other dilutions were prepared from this first 1021 dilution to dilutions of 1026. MRS agar (Merck, Darmstadt, Germany), L-cysteine hydrochloride monohydrate and mupirocin were used for the enumeration of B. bifidum. The agar plates were incubated anaerobically at 35–37C at least for 72 h. L. acidophilus in the yogurt samples was also
Preparation of Barberry Extract The extraction process was carried out using a reflux system set up in a water bath. Then, 200 g of barberry fruits were 2
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FIG. 1. VIABLE COUNTS OF L. ACIDOPHILUS STRAIN (106 CFU/GR) IN THE PROBIOTIC SET AND STIRRED YOGURTS CONTAINING OF BARBERRY EXTRACT DURING REFRIGERATED STORAGE (Sample (1) control yoghurt sample, sample (2) set yoghurt sample containing 4% of barberry extract, sample (3) set yogurt sample containing 5% of barberry extract, sample (4) stirred yogurt sample containing 4% of barberry extract, sample (5) stirred yogurt sample containing 5% of barberry extract).
cultured using MRS agar in aerobic conditions at 35–37C for 72 h (Vinderola and Reinheimer 1999). Direct counting was done through the use of an automatic colony counter. Microbiological analyses were carried out in triplicate and the results were reported in terms of cfu/gr (colony-forming units per milliliter).
Statistical Analysis A completely randomized factorial design was selected and the experiments were performed in at least triplicate. The data were then submitted to Statistical Analysis Systems (SAS, 9.1.3, USA) and finally, Duncan’s multiple range test was run to assess any statistically significant difference between the obtained mean values in each experiment at P
