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The comparative assessment of ACE-inhibitory and antioxidant activities of peptide fractions obtained from fermented camel and bovine milk by Lactobacillus rhamnosus PTCC 1637
Accepted Manuscript The comparative assessment of ACE-inhibitory and antioxidant activities of peptide fractions obtained from fermented camel and bovine milk by Lactobacillus rhamnosus PTCC 1637 Maryam Moslehishad, Mohammad Reza Ehsani, Maryam Salami, Saeed Mirdamadi, Hamid Ezzatpanah, Amir Niasari Naslaji, Ali Akbar Moosavi-Movahedi PII:
S0958-6946(12)00244-0
DOI:
10.1016/j.idairyj.2012.10.015
Reference:
INDA 3443
To appear in:
International Dairy Journal
Received Date: 7 July 2012 Revised Date:
14 October 2012
Accepted Date: 29 October 2012
Please cite this article as: Moslehishad, M., Ehsani, M.R., Salami, M., Mirdamadi, S., Ezzatpanah, H., Naslaji, A.N., Moosavi-Movahedi, A.A., The comparative assessment of ACE-inhibitory and antioxidant activities of peptide fractions obtained from fermented camel and bovine milk by Lactobacillus rhamnosus PTCC 1637, International Dairy Journal (2012), doi: 10.1016/j.idairyj.2012.10.015. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
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The comparative assessment of ACE-inhibitory and antioxidant 1 activities of peptide fractions obtained from fermented camel and bovine2 milk by Lactobacillus rhamnosus PTCC 1637
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c Maryam Moslehishada, Mohammad Reza Ehsania٭, Maryam Salamib, Saeed Mirdamadi 5 ,
Hamid Ezzatpanaha, Amir Niasari Naslajid, Ali Akbar Moosavi-Movahedib,e† 6
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Food Science and Technology Department, Faculty of Agriculture and Natural 9 Resources,
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Science and Research Branch, Islamic Azad University, Tehran, Iran.
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Institute of Biochemistry and Biophysics, University of Tehran, Tehran, Iran. 11
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Iranian Research Organization for Science & Technology (IROST), Dept. 12 of Biotechnology,
Tehran, Iran.
Department of Clinical Sciences, Faculty of Veterinary Medicine, University 14 of Tehran,
Tehran, Iran.
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Center of Excellence in Biothermodynamics, University of Tehran, Tehran, 16Iran 17
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*†Corresponding authors. *Tel.: +98 21 66403957; †Tel.: +98 21 4486513721 E-mail addresses: [email protected] (M. R. Ehsani) [email protected] (A. 22 A. MoosaviMovahedi)
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__________________________________________________________________________ 25 Abstract
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The aim of this study was to compare angiotensin converting enzyme28(ACE)inhibitory and antioxidant activities of peptide fractions obtained from fermented 29 bovine and camel milk by Lactobacillus rhamnosus PTCC 1637 during 21 days of cold30 storage. The
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proteolytic activity was determined using the o-phthaldialdehyde method, antihypertensive 31 effect was performed based on inhibition of ACE activity, and antioxidant activity 32 was
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measured using the 2,2'-azino-bis-(3-ethyl-benzthiazoline-6-sulfonic acid) scavenging 33 assay. In most cases, higher ACE-inhibitory and antioxidant activity was observed34 from cultured camel milk than bovine milk. This may be explained by structural differences 35 and the presence of higher proline content in the primary structure of camel milk caseins 36 compared with bovine milk. In both milk types, increased proteolytic activity during storage 37 resulted in
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increased antioxidant activity. The results suggest the potential use of fermented 38 camel milk with Lb. rhamnosus PTCC 1637 for production of dairy product with ACE-inhibitory 39 and antioxidant properties.
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1. Introduction
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Milk proteins possess numerous biological activities that make these54 components
effective in improving human health and nutrition (Park, 2009). In recent years, 55 it has been
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recognised that not only intact caseins and whey proteins but also many milk 56protein-derived peptides exhibit physiological functions (Clare & Swaisgood, 2000; Haquea, 57Chanda, &
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Kapilab, 2009). Bioactive peptides can be released from milk protein sequences 58 by digestive proteases, microbial or plant enzymes or by fermentation using dairy starter59 cultures with proteolytic activities (Hayes, Ross, Fitzgerald, & Stanton, 2007; Korhonen & 60Pihlanto, 2006). These peptides reveal various biological properties including antimicrobial 61 (López-Expósito & Recio, 2006), cholesterol-lowering (Hartmann & Meisel, 2007), mineral-binding 62 (Vegarud,
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Langsrud, & Svenning, 2000), immunomodulatory, opioid, antioxidative and 63 antihypertensive effects (Jäkälä & Vapaatalo, 2010; Korhonen, 2009).
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The main bioactive peptides studied are those with antihypertensive 65 effect (Korhonen
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& Pihlanto, 2006), which act as inhibitors of angiotensin-converting enzyme66(ACE). Inhibition of ACE plays an important role in regulation of blood pressure (De 67 Leo, Panarese,
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Gallerani, & Ceci, 2009). Milk-derived ACE inhibitory peptides inhibit conversion 68 of angiotensin I to angiotensin II; therefore they have a blood pressure lowering 69effect (Vermeirssen, Van Camp, & Verstraete, 2004). These peptides have already70been isolated from a variety of fermented dairy products including cheese (Hartmann & Meisel, 71 2007), yoghurt (Donkor, 2007) and fermented bovine milk (Qian et al., 2011).
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Recently, interest in food-derived antioxidant peptides and evidence73 that these peptides prevent oxidative stress has increased (Xiong, 2010; Zhao, Wu, & 74 Li, 2010).
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Oxidation of lipids is one of the major causes of food deterioration and shelf75life reduction in the food industry (Pihlanto, 2006). Moreover, lipid oxidation can generate free 76 radicals that play a significant role in cardiovascular disease (Maxwell & Lip, 1997), Alzheimer’s 77 disease
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(Sonnen et al., 2009) and certain cancers (Blot et al., 1993). Enzymatic and 78 non-enzymatic antioxidants such as superoxide dismutase, catalase and α- tocopherol protect 79 cells from
oxidative damage and provide a range of health-promoting benefits (Xiong,80 2010). In recent
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years, some of the milk protein-derived bioactive peptides have been considered 81 as a novel class of antioxidants (Pihlanto, 2006).
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Milk fermentation by proteolytic lactic acid bacteria (LAB) is one of83the economical and practical methods for the production of fermented dairy products enriched 84 in bioactive peptides (Hayes et al., 2007). It has been previously reported that bovine milk 85 fermented with proteolytic strains of LAB such as Lactobacillus helveticus JCM1004 (Pan, 86 Luo, & Tanokura, 2005), Lactobacillus delbrueckii subsp. bulgaricus (Papadimitriou 87et al., 2007),
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Lactococcus lactis YT2027 and Lactobacillus acidophilus DPC6026 contain88health-beneficial bioactive peptides (Fitzgerald & Murray, 2006; Hayes, Ross, Fitzgerald, Hill, 89 & Stanton, 2006).
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Milk from different species (bovine, goat, sheep, buffalo, etc.) is used 91for production of fermented milk products (Chandan, 2004). Camel milk for fermented milk 92production
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contains well-balanced nutrients and biological components. Camel milk is 93 high in vitamin C and niacin, as well as richer in Cu and Fe than bovine milk (El-Agamy, 2006). 94 Moreover, camel milk lacks β-lactoglobulin and contains α-lactalbumin, a similar situation 95 to that in human milk. Previous research has recommended the potential use of camel96milk in the manufacture of infant formula (Salami et al., 2009). The antioxidant and ACE-inhibitory 97 property of casein-derived peptides of camel milk by digestive proteases has98already been studied (Salami et al., 2011). The significantly higher digestibility and greater 99 antioxidant
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activity of camel milk α-lactalbumin compared with that of bovine whey 100 proteins was also observed in a previous study (Salami et al., 2009). Furthermore, some reports 101 are available on peptides with a wide range of functionalities in fermented bovine milk with 102proteolytic
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strains of LAB (Fitzgerald & Murray, 2006; Hayes et al., 2007; Papadimitriou 103 et al., 2007), but there is a lack of information in the literature about biological activity104 of camel milk-
derived peptides by lactic acid fermentation. Therefore, it would be interesting 105 to investigate
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the functionalities of bioactive peptides produced during the fermentation106 of camel milk.
Based on our previous study, Lactobacillus rhamnosus PTCC 1637 107 was selected for
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this study due to its high protease activity and high mean score values for108 sensory quality in fermented bovine and camel milk (Moslehishad, Mirdamadi, Ehsani, Moosavi-Movahedi, 109 & Ezzatpanah, 2012). The objective of this study was to compare the ACE-inhibitory 110 and antioxidant activity of peptide fractions obtained from camel and bovine milk 111 fermented with
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Lb. rhamnosus PTCC 1637 during 21 days of cold storage.
Materials and methods
Milk collection
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2.1.
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Bovine milk samples were obtained from a commercial dairy farm118 in Tehran province, Iran. Camel milk samples were collected from local camel producers 119 in Torkaman Sahra, Gorgan province, Iran. All milk samples were stored at 4 °C during120 transportation to the laboratory.
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Milk fermentation
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Lb. rhamnosus PTCC 1637 was obtained from Persian Type Culture 125Collection (PTCC, Tehran, Iran). It was maintained at -70 ˚C in 15% skim milk (Merck, 126 Darmstadt, Germany) plus 30% glycerol (Merck) before further experiments. The organism 127 was
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activated in sterile 10 mL aliquots of de Man Rogosa and Sharpe (MRS) broth 128 (Merck) at 37 ºC for 24 h. The cultures were centrifuged at 5000 × g for 15 min to separate 129 bacteria and
were washed twice with sterile distilled water. Biomass was then inoculated 130into skim milk
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12% (w/v) and incubated at 37 ºC for 24 h as pre-culture to obtain approximately 131 108 cfu mL. Fresh whole bovine and camel milk samples were pasteurised at 80 ºC for 13220 min in a
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water bath and cooled to 43 ºC. The pre-cultures were then inoculated (2%, 133v/v) into bovine and camel milk and incubated at 37 ºC for 24 h. Fermented milk samples 134 were stored at 5 ± 1 ºC for 21 days. All analyses were carried out at days 1, 7, 14 and 21 of storage. 135 The data are the mean values of two independent experiments (batches) assayed in triplicates. 136
Bacterial counts
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Cell population of Lb. rhamnosus was determined by counting colony-forming 140 units
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(cfu) on MRS agar after incubation at 37 °C for 48 h in candle jar under enriched 141 CO2 (Matalon & Sandine, 1986).
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2.4.
Determination of proteolytic activity
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Fermented milk (2.5 mL) was added to 5 mL of 0.75% trichloroacetic 146 acid (TCA) and
filtered through filter paper (Whatman No. 2). Proteolytic activity of filtrate 147was then measured at 340 nm in a spectrophotometer (Shimadzu, model UV-3100,148 Kyoto, Japan) as
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previously described by Church, Swaisgood, Porter, and Catignani (1983)149 and Donkor (2007).
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Preparation of peptide fractions
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2.5.
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The water-soluble extract (WSE) of the fermented bovine and camel 154milk was
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separated by centrifugation at 12,000 × g for 10 min at 4 ◦C and by filtration 155 through
Whatman no. 40 filter (Quiros, Hernandez-Ledesma, Ramos, Amigo, & Recio, 156 2005). The
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WSE was fractioned using ultrafiltration (UF) membranes with molecular157 mass cut off sizes of 10, 5 and 3 kDa (Amicon Ultra-15, Millipore, Carrigtwahill, Co. Cork,158 Ireland). The permeates and retentate of each stage of filtration were collected and stored 159at -20 °C until further experiments.
Measurement of ACE-inhibitory activity
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ACE-inhibitory activity of peptide fractions of fermented milk samples 164 were
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determined by the method of Vermeirssen, Van Camp, and Verstraete (2002) 165 and Salami et al. (2011). Rabbit lung extract (Sigma-Aldrich, Munich, Germany) as the166 source of ACE was
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prepared by dissolving 1 g of rabbit lung acetone powder in 10 mL of 50 167 mM potassium phosphate buffer (pH 8.3) and 5% (v/v) glycerol. The mixture was centrifuged 168 for 40 min at 40,000 × g after overnight storage at 4 °C. The supernatant was separated169 and stored at 4 °C before analysis. An aliquot (150µL) of furanacrylolyl tripeptide (1mM) (Sigma-Aldrich) 170 was dissolved in 50 mM Tris-HCl buffer, pH 8.3, containing 400 mM NaCl, peptide 171 fractions were added to each well of ELISA plates. ACE extract was added to the mixture 172and pre-incubated at 37 °C for 2 min. The absorbance was measured at 340 nm using an ELISA 173 reader Expert
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96 (ASYS Hi-tech, Eugendorf, Austria) for periods of 25 min. The ACE activity 174 (unit L-1) of each peptide fraction was calculated according to the below formula:
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(1)
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ACE activity = Vt. 1,000. ∆A ∆e. Vs . d
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where Vt = total volume; Vs= ACE volume; d = light path (cm); ∆e = maximum 180 change of absorbance at 340 nm; and ∆A = absorbance changes per minute. Subsequently, 181 ACE
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inhibition was reported as IC50 (inhibitory concentration 50%) values.
Determination of antioxidant activity
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Antioxidant activity of peptide fractions of fermented milk samples 186was evaluated by
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Trolox Equivalent Antioxidant Capacity (TEAC) method based on scavenging 187 of the 2,2'azino-bis-(3-ethyl-benzthiazoline-6-sulfonic acid) (ABTS; Sigma-Aldrich) 188 as described previously by Re et al. (1999) and Salami et al. (2011). ABTS assay was 189 carried out by
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oxidising ABTS (7 mM) in 2.45 mM potassium persulphate buffer for 12–16 190 h in the dark to provoke the formation of ABTS radical. ABTS•+ solution was diluted in 5191 mM phosphate
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buffer, pH 7.4, to obtain absorbance 0.7 ± 0.2 at 734 nm. ABTS•+ solution, 192 reactive towards antioxidant compounds, was added to peptide fractions. The mixture was193 incubated at 25 °C for 5 min and the absorbance was measured at 734 nm using an ELISA reader 194 Expert 96. A 6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid (Trolox) (Sigma-Aldrich) 195 standard curve was used to determine Trolox equivalent values.
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2.8.
Statistical analysis
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All data were subjected to one-way analysis of variance (ANOVA) 200 using SPSS 12.0 software (SPSS INC., Chicago, IL, USA, 2002). Significant treatment means 201 were separated by Duncan’s New Multiple Range Test.
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3.
Results and Discussion
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3.1.
Cell growth during fermentation and storage
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Viable counts of Lb. rhamnosus PTCC 1637 after fermentation and 208 during cold storage time are shown in Fig. 1. The bacterial counts for fermented bovine 209and camel milk ranged initially between 8.14 to 8.86 and 8.54 to 8.90 log cfu mL-1 after fermentation, 210 respectively. In both milk types, the counts did not change significantly (P 211≥ 0.05) until day
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21 and were in the range of 8.19 to 8.40 and 8.71 to 8.80 log cfu mL-1 for212 bovine and camel cultured milk, respectively. The slow growth rate of bacterial cells during213 cold storage could be due to the preference of mesophilic to slightly thermophilic temperature 214for LAB
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(Hammes & Hertel, 2009). Moreover, the release of peptides and amino acids 215 as the nutrient supply by the proteolytic system of Lb. rhamnosus may explain the viability 216 and minimal
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decline in cell population (Pastar et al., 2003; Savijoki, Ingmer, & Varmanen, 217 2006). No significant differences (P ≥ 0.05) were found in the growth of 218 Lb. rhamnosus
between camel and bovine milk products. Contradictory results have been219 published about the growth and viability of strains used as starters in camel milk compared 220 with bovine milk. Abu-Tarboush (1996) stated that Streptococcus thermophilus and Lb. delbrueckii 221 ssp. bulgaricus grow at a faster rate in bovine milk than in camel milk. Abu-Tarboush, 222 Al-Dagal, and AL-Royl (1998) reported faster growth rate of Bifidobacterium longum 22315707 and slower
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cell growth of Bifidobacterium angulatum 27535 in camel milk than in bovine 224 milk. This probably demonstrates that the type of strain is the determinant factor that225 influences the bacterial growth rate of organisms in milk from different species.
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Proteolysis in fermented milk
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Proteolytic activity of fermented milk was assessed during cold storage 230 based on
determination of peptide release (Fig. 2). Milk fermentation leads to formation 231 of peptides as
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the growth factor for LAB and an excess amount of peptides can be accumulated 232 in the medium (Donkor, 2007). These peptides were measured using o-phthaldialdehyde 233 (OPA) spectrophotometric assay after fermentation and during prolonged cold storage. 234 As expected, the OPA values (extent of proteolysis) increased significantly (P < 0.05) in 235both fermented bovine and camel milk until day 21. This could probably be due to the survival 236 of
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microorganisms in the presence of free NH3 groups in the medium by the237 end of storage. Furthermore, the release of cell-wall attached proteinase may occur after 238 the lysis of bacteria during shelf life (Ramchandran, 2009). The data are consistent with those239 reported by
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Nielsen, Petersen, and Dambmann (2001) and Donkor (2007). In the present 240 study, the increased proteolysis of fermented milk by Lb. rhamnosus PTCC 1637 until 241the end of
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storage, suggests that it is one of the proteolytic strains of LAB as our pervious 242 findings also revealed the same result (Moslehishad et al., 2012). Data showed that approximately 243 similar trends of proteolytic activity were obtained in fermented bovine and camel 244 milk. Our results differ from those reported by Abu-Tarboush (1996), who found higher proteolysis 245 of yoghurt starters in camel milk than in bovine milk. However, our findings might be 246due to straindependent differences.
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3.3.
ACE-inhibitory activity of peptide fractions of fermented milk
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In the present study, in vitro ACE-inhibitory activity of WSE to identify 251 the biological
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activity of cultured milk samples and peptide fractions from fermented bovine 252 and camel milk separated by UF membranes with molecular mass cutoff of 10, 5 and253 3 kDa were
determined (Table 1). The ACE-inhibitory activity of milk protein-derived 254 peptides was
determined as IC50 value based on inhibiting ACE activity (Haquea et al. 255 2009). As shown in
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Table 1, the lowest IC50 value was 1.45±0.02 mg mL-1. The IC50 value was 256 considerably
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lower than that reported by Qian et al. (2011) for the most potent ACE-inhibitory 257 peptide fraction obtained from fermented bovine milk by Lb. delbrueckii ssp. bulgaricus 258 LB with an IC50 value of 67.71±7.62 mg mL-1. This may suggest that ACE-inhibitory259 peptide formation is related to the bacterial strains and culture conditions used in the fermentation 260 process. Furthermore, Ferreira et al. (2007) reported that amino acid residues/sequences 261
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greatly contribute to ACE-inhibitory potency of bioactive peptides. Therefore, 262 the differences in the primary structure of camel milk proteins than in bovine milk (Kappeler, 263 Farah, & Puhan, 1998) may also influence the ACE-inhibitory activity of camel milk 264peptide fractions.
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Table 1 also shows that in the case of both bovine and camel milk,265 the lowest IC50 values were observed in the <5 kDa peptide fractions obtained after hydrolysis 266 of milk
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proteins with proteolytic enzymes of Lb. rhamnosus. Generally, most of the 267demonstrated antihypertensive peptides are short peptides with a Pro residue at the C-terminus 268 (Mizuno, Nishimura, Matsuura, Gotou, & Yamamoto, 2004). Short peptides containing 269 Pro at the Cterminal end are resistant against digestive enzymes and more readily transfer 270 to the blood stream (Korhonen & Pihlanto, 2006). Gobbetti, Ferranti, Smacchi, Goffeedi, 271 and Addeo (2000) reported the formation of ACE-inhibitory peptides released from milk 272 proteins by starter cultures containing proline-specific peptidases during fermentation. 273 Lb. rhamnosus
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possesses a proteolytic system that includes a proline-specific peptidase like 274 proline-specific aminopeptidase (PepR) and X-prolyl-dipeptidyl aminopeptidase (PepX; Pastar 275 et al., 2003; Savijoki et al., 2006) that subsequently may result in accumulation of bioactive 276 ACE-
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inhibitors in fermented milk. This may explain the results obtained in this277 work which indicates that Lb. rhamnosus PTCC 1637 is capable of producing potent ACE-inhibitory 278
peptides from bovine and camel milk proteins, especially from caseins containing 279 a high level
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of proline residues. These results are in agreement with those obtained by280 Hernández-
Ledesma, Amigo, Ramos, and Recio (2004) when studying ACE-inhibitory 281activity of bovine
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milk fermented with Lb. rhamnosus CECT 287T. They also reported the release 282 of ACEinhibitory activity peptides during fermentation of bovine milk.
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The ACE-inhibitory values of WSE found for cultured bovine milk 284 were in the range of 2.483±0.148 to 3.947±0.029 mg mL-1 and 2.223±0.052 to 3.930±0.118285 mg mL-1 for camel milk during 21 days of storage. The results indicated that the IC50 values,286 especially in camel
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milk, were considerably lower than that reported by Chen et al. (2010) for287 koumiss whey (52.47±2.87 mg mL-1). Moreover, in this study higher ACE-inhibitory activity 288 was observed from cultured camel milk peptide fractions than bovine milk counterparts.289 This may be
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explained by the presence of higher proline content in the primary structure 290of camel milk caseins than in bovine milk (El-Agamy, 2006). Our results were in agreement 291 with those
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reported by Salami et al. (2011) that showed a significant antihypertensive 292 property of camel milk casein following enzymatic digestion.
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The extent of ACE inhibition did not follow any specific trend during 294 21 days of
storage at 5 °C (Table 1). This finding suggests that some of the peptides 295 produced at the early or intermediate stage of hydrolysis may degrade and new ones may 296 be produced due to the secondary proteolysis throughout the shelf life in both fermented milk297 samples. Similar trends were previously reported by Donkor (2007). In contrast, Nielsen, Martinussen, 298
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Flambard, Sørensen, and Otteet (2009) found that the ACE-inhibitory activity 299 of fermented milk using Lc. lactis and Lb. helveticus strains 1198 and 1263 increased during 300 7 days of cold storage. Consequently, the different results on the trends of ACE-inhibition 301during storage
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period may be explained by the significant effect of bacterial strains on hypotensive 302 peptide production during refrigerated storage time in fermented milk products. 303 304
3.4.
Determination of antioxidant activity
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The antioxidant activity of peptide fractions (5-10, 3-5, and < 3 kDa) 307 and WSE of
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fermented bovine and camel milk by Lb. rhamnosus showed that fermentation 308 increased the ABTS radical scavenging activity (Figs 3 and 4). This may be due to the hydrolysis 309 of αS1and β-casein by proteolytic/peptidolytic enzymes of Lb. rhamnosus (Varmanen, 310 Rantanen, Palva, & Tynkkynen, 1998; Pastar et al., 2003). Moreover, production of 311 PrtR proteinase,
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PepO endopeptidase (NH2–Xn↓Xn–COOH), PepR (NH2–Pro↓X–COOH)312 and PepX (NH2– X–Pro↓Xn–COOH) by Lb. rhamnosus has been previously reported by Pastar 313 et al. (2003) and Savijoki et al. (2006).
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The 5-10 kDa peptide fractions exhibit the highest radical scavenging 315 activities compared with lower molecular masses of peptides in both fermented milk. 316The level of
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TEAC values of 5-10 kDa peptides were in the range of 110.41 to 745.35317 µM and 844.08 to 1737.88 µM in fermented bovine and camel milk, respectively (Figs. 3 and 318 4). Peptide fractions extracted from camel milk fermented by Lb. rhamnosus showed319 higher antioxidant activity than in bovine milk. The findings suggest that not only peptide size, 320but also the nature and composition of peptides, which is not similar in fermented bovine 321 and camel milk, plays an important role in quenching of ABTS radicals and antioxidant efficiency. 322 The presence of hydrophobic and aromatic amino acid residues in323 peptides is a determinant factor in radical scavenging (Ajibola, Fashakin, Fagbemi, & 324 Aluko, 2011). 13
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Certain amino acids such as His (imidazole group), Trp (indolic group), Tyr 325(phenolic group), Phe (aromatic amino acid) and Pro (hydroxyl radical scavenger) correlated 326 with antioxidant capacity. Furthermore, Met and Cys are effective in quenching of free radicals 327 due to their
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ability to donate sulphur hydrogen (Ajibola et al., 2011; Xiong, 2010). It was 328 hypothesised that the higher level of Pro, Met, Ile, Trp residues in camel caseins than in329 bovine milk leads to an increase in free-radical scavenging activity of peptide fractions extracted 330 from
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fermented camel milk compared with bovine milk samples (El-Agamy, 2006). 331
In the current study, the WSE of both fermented milk products showed 332 significantly
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(P < 0.05) the highest antioxidant activity compared with peptide fractions 333 (Figs 3 and 4). The TEAC values ranged from 277.35 to 930.41 µM and 1417.88 to 2035.61 334 µM in fermented bovine and camel milk, respectively. This may be due to the synergistic effect 335 between peptides and milk proteins enhancing antioxidant capacity (Kwak, Seo, &336 Lee, 2009). Radical scavenging capacity of milk proteins has been previously reported 337 by Chiang and
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Chang (2005). Furthermore, Salami et al. (2010) have reported higher antioxidative 338 activity of camel whey proteins than bovine whey, which is in accordance with our 339 results, where more pronounced ABTS radical scavenging activity of WSE of fermented340 camel milk by Lb. rhamnosus than bovine milk was observed.
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As shown in Fig.3, the highest TEAC value (2053.61 µM) was observed 342 in the WSE
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of fermented camel milk by Lb. rhamnosus on day 21. This result is considerably 343 higher than that reported previously by Virtanen, Pihlanto, Akkanen, and Korhonen (2007) 344 for Leuconostoc mesenteroides ssp.cremoris (700 µM) and Lactobacillus jensenii 345 (610 µM). This may suggest that the development of antioxidative activity during milk fermentation 346 is a strain-dependent factor. Compared with protease digestion by trypsin and347 papain, as previously reported by Pattorn & Hongsrragas (2012) and Zhao et al. (2010) 348 respectively, milk fermentation using Lb. rhamnosus, especially in camel milk, revealed 349 higher scavenging
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activity. The results may suggest the potential use of fermentation in enhancing 350 natural antioxidant factors of bovine and camel milk products.
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In the present study, the antioxidant capacity of fermented bovine352 and camel milk
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products did not decline at the end of the storage period. Noticeably, the extent 353 of TEAC values in both bovine and camel milk after fermentation and at weekly intervals 354 was timedependent and the highest value (P < 0.05) was observed on day 21 (Figs355 3 and 4). This
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finding may be due to the presence of vital microorganisms and the release 356of enzymes from them within the food matrix during storage time. The data obtained in this357 study showed the
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increased proteolysis and survival of Lb. rhamnosus during cold storage which 358 may result in high level of antioxidant activity at the end of the storage period. Our finding 359 was also in agreement with those reported by Virtanen et al. (2007) which revealed the 360relationship between the development of antioxidant activity and a high degree of proteolysis. 361
Conclusion
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Milk fermentation resulted in the release of peptides as the growth365 factor for Lb.
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rhamnosus. Fermentation of milk by Lb. rhamnosus as a proteolytic strain366 can cause to release ACE-inhibitory and antioxidant peptides from bovine and camel milk 367 proteins. This
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work also confirms that the enhancement of proteolytic activity contributed 368to the increased antioxidant activity of both fermented milk types during cold storage. ACE-inhibitory 369 and antioxidant activity of fermented camel milk by Lb. rhamnosus were more 370 pronounced than bovine milk. A potential for using these findings is the development of fermented 371 camel milk by LAB as a novel food product containing ACE-inhibitory and antioxidant 372peptides. 373
Acknowledgements
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The support of the University of Tehran, Iranian Research Organization 376 for Science and Technology (IROST), Iran National Science Foundation (INSF), Iran377 Dairy Industries
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Company (Pegah) and Islamic Azad University, Science and Research Branch 378 of Tehran are gratefully acknowledged.
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Abu-Tarboush, H. M. (1996). Comparison of associative growth and proteolytic 383 activity of yogurt starters in whole milk from camels and cows. Journal of Dairy 384 Science, 79, 366–371.
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Abu-Tarboush, H. M., Al-Dagal, M. M., & AL-Royl, M. A. (1998). Growth, 386 viability, and proteolytic activity of Bifidobacteria in whole camel milk. Journal387 of Dairy Science, 388
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81, 354–361.
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Table 1
1
PTCC 1637 during cold storage. a
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ACE inhibitory activities (IC50) of peptide fractions and water-soluble extract2 of fermented camel and bovine milk by Lactobacillus rhamnosus 3 4
Storage Fraction size (kDa) time (d) 5-10 3-5
Water soluble extract
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Fermented milk
<3
1
1.790±0.010 aA
2.545±0.185 bA
1.830±0.160 acA
3.930±0.118dA
Bovine
1
2.490±0.040 aB
3.810±0.175 bB
1.510±0.030 cA
3.947±0.029 bA
Camel
7
4.475±0.045 aA
1.450±0.015 bA
2.960±0.040 cA
2.423±0.200 dA
Bovine
7
4.200±0.006 aA
2.040±0.060 bB
3.840±0.073cB
3.653±0.045 cB
Camel
14
3.405±0.205 aA
3.500±0.050 aA
2.250±0.077 bA
2.873±0.094 cA
Bovine
14
4.325±0.005 aA
1.510±0. 175bB
2.183±0.024 cA
2.483±0.148 cA
Camel
21
2.210±0.064 aA
2.310±0.036 aA
3.710±0.020 bA
2.223±0.052 aA
Bovine
21
2.830±0.082 aB
1.803±0.095 cB
3.223±0.187 aB
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4.095±0.195 bB
5
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IC50 is defined as the concentration (mg mL-1) required to inhibit 50% of ACE 6 activity. Data are means ± standard error of the mean of triplicate measurements of two independent experiments (batches); values with7 different superscript lower case letters in a row and with different superscript upper case letters in a column are statistically significant at P < 0.05. 8 9
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a
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Camel
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Cell population (log cfu mL-1)
a/A a/A
a/A
a/A
a/A
a/A
a/A
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a/B
Fermented bovine milk
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Fermented camel milk
1
2 3 4
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1 2 3
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4
d
c
bc
a
b
b
a
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a
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c
b
1
5 6
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1
c/C
c/B
TEAC (µM)
d/A
a/A
a/AB a/C
a/C
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c/B
b/C
a/B
b/D
b/A
b/C c/A
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b/B b/B
1
2 3
4
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TEAC (µM)
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1 2
c/C
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a/B
c/B b/AB
b/C b/C
a/A a/A a/A
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c/A
a/A
a/A
a/A
b/B
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b/B b/B
3 4
5
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Figure legends
1 2
Fig. 1. Population of bacteria in fermented bovine and camel milks 3with Lactobacillus ), 7 (
), 14 ( ),421( ). Error bars
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rhamnosus PTCC 1637 during cold storage at days 1(
show standard error of the mean of triplicate measurements of two independent 5 experiments (batches). Mean values with different small letters for the same fermented6 milk type and in
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capital letters for the same storage time are significantly different (P < 0.05). 7 8
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Fig. 2. Proteolytic activity of fermented bovine () and camel () milks 9with Lactobacillus rhamnosus PTCC 1637 during cold storage. Error bars show standard error 10 of the mean of triplicate measurements of two independent experiments (batches). Different 11 letters indicate
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significant differences in fermented milk during cold storage (P < 0.05).
Fig. 3. Antioxidant activities of peptide fractions 5-10 kDa (
12 13
), 3-5 kDa (14 ), <3 kDa (
) and
water-soluble extract (WSE) ( ) of fermented camel milk with Lactobacillus 15 rhamnosus PTCC
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1637 during storage. The data measured as Trolox equivalent antioxidant16 capacity (µ M). Error bars show standard error of the mean of triplicate measurements 17 of two independent
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experiments. Mean values with different small letters for the same storage 18 time and in capital letters for the same molecular mass peptide fraction or WSE during cold storage 19 are significantly different (P < 0.05).
20 21
Fig. 4. Antioxidant activities of peptide fractions 5-10 kDa ( water-soluble extract (WSE) (
), 3-5 kDa (22 ), <3 kDa (
) and
) of fermented bovine milk with Lactobacillus 23 rhamnosus PTCC
1637 during cold storage. The data measured as Trolox equivalent antioxidant 24 capacity (µ M).
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Error bars show standard error of the mean of triplicate measurements25of two independent experiments (batches). Mean values with different small letters for the same 26 storage time and in capital letters for the same molecular mass peptide fraction or WSE during 27 cold storage are significantly different (P < 0.05).
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S0958-6946(12)00244-0
DOI:
10.1016/j.idairyj.2012.10.015
Reference:
INDA 3443
To appear in:
International Dairy Journal
Received Date: 7 July 2012 Revised Date:
14 October 2012
Accepted Date: 29 October 2012
Please cite this article as: Moslehishad, M., Ehsani, M.R., Salami, M., Mirdamadi, S., Ezzatpanah, H., Naslaji, A.N., Moosavi-Movahedi, A.A., The comparative assessment of ACE-inhibitory and antioxidant activities of peptide fractions obtained from fermented camel and bovine milk by Lactobacillus rhamnosus PTCC 1637, International Dairy Journal (2012), doi: 10.1016/j.idairyj.2012.10.015. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
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The comparative assessment of ACE-inhibitory and antioxidant 1 activities of peptide fractions obtained from fermented camel and bovine2 milk by Lactobacillus rhamnosus PTCC 1637
3
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4
c Maryam Moslehishada, Mohammad Reza Ehsania٭, Maryam Salamib, Saeed Mirdamadi 5 ,
Hamid Ezzatpanaha, Amir Niasari Naslajid, Ali Akbar Moosavi-Movahedib,e† 6
8
Food Science and Technology Department, Faculty of Agriculture and Natural 9 Resources,
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a
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Science and Research Branch, Islamic Azad University, Tehran, Iran.
10
b
Institute of Biochemistry and Biophysics, University of Tehran, Tehran, Iran. 11
c
Iranian Research Organization for Science & Technology (IROST), Dept. 12 of Biotechnology,
Tehran, Iran.
Department of Clinical Sciences, Faculty of Veterinary Medicine, University 14 of Tehran,
Tehran, Iran.
15
Center of Excellence in Biothermodynamics, University of Tehran, Tehran, 16Iran 17
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*†Corresponding authors. *Tel.: +98 21 66403957; †Tel.: +98 21 4486513721 E-mail addresses: [email protected] (M. R. Ehsani) [email protected] (A. 22 A. MoosaviMovahedi)
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__________________________________________________________________________ 25 Abstract
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The aim of this study was to compare angiotensin converting enzyme28(ACE)inhibitory and antioxidant activities of peptide fractions obtained from fermented 29 bovine and camel milk by Lactobacillus rhamnosus PTCC 1637 during 21 days of cold30 storage. The
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proteolytic activity was determined using the o-phthaldialdehyde method, antihypertensive 31 effect was performed based on inhibition of ACE activity, and antioxidant activity 32 was
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measured using the 2,2'-azino-bis-(3-ethyl-benzthiazoline-6-sulfonic acid) scavenging 33 assay. In most cases, higher ACE-inhibitory and antioxidant activity was observed34 from cultured camel milk than bovine milk. This may be explained by structural differences 35 and the presence of higher proline content in the primary structure of camel milk caseins 36 compared with bovine milk. In both milk types, increased proteolytic activity during storage 37 resulted in
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increased antioxidant activity. The results suggest the potential use of fermented 38 camel milk with Lb. rhamnosus PTCC 1637 for production of dairy product with ACE-inhibitory 39 and antioxidant properties.
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1. Introduction
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53
Milk proteins possess numerous biological activities that make these54 components
effective in improving human health and nutrition (Park, 2009). In recent years, 55 it has been
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recognised that not only intact caseins and whey proteins but also many milk 56protein-derived peptides exhibit physiological functions (Clare & Swaisgood, 2000; Haquea, 57Chanda, &
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Kapilab, 2009). Bioactive peptides can be released from milk protein sequences 58 by digestive proteases, microbial or plant enzymes or by fermentation using dairy starter59 cultures with proteolytic activities (Hayes, Ross, Fitzgerald, & Stanton, 2007; Korhonen & 60Pihlanto, 2006). These peptides reveal various biological properties including antimicrobial 61 (López-Expósito & Recio, 2006), cholesterol-lowering (Hartmann & Meisel, 2007), mineral-binding 62 (Vegarud,
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Langsrud, & Svenning, 2000), immunomodulatory, opioid, antioxidative and 63 antihypertensive effects (Jäkälä & Vapaatalo, 2010; Korhonen, 2009).
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The main bioactive peptides studied are those with antihypertensive 65 effect (Korhonen
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& Pihlanto, 2006), which act as inhibitors of angiotensin-converting enzyme66(ACE). Inhibition of ACE plays an important role in regulation of blood pressure (De 67 Leo, Panarese,
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Gallerani, & Ceci, 2009). Milk-derived ACE inhibitory peptides inhibit conversion 68 of angiotensin I to angiotensin II; therefore they have a blood pressure lowering 69effect (Vermeirssen, Van Camp, & Verstraete, 2004). These peptides have already70been isolated from a variety of fermented dairy products including cheese (Hartmann & Meisel, 71 2007), yoghurt (Donkor, 2007) and fermented bovine milk (Qian et al., 2011).
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Recently, interest in food-derived antioxidant peptides and evidence73 that these peptides prevent oxidative stress has increased (Xiong, 2010; Zhao, Wu, & 74 Li, 2010).
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Oxidation of lipids is one of the major causes of food deterioration and shelf75life reduction in the food industry (Pihlanto, 2006). Moreover, lipid oxidation can generate free 76 radicals that play a significant role in cardiovascular disease (Maxwell & Lip, 1997), Alzheimer’s 77 disease
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(Sonnen et al., 2009) and certain cancers (Blot et al., 1993). Enzymatic and 78 non-enzymatic antioxidants such as superoxide dismutase, catalase and α- tocopherol protect 79 cells from
oxidative damage and provide a range of health-promoting benefits (Xiong,80 2010). In recent
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years, some of the milk protein-derived bioactive peptides have been considered 81 as a novel class of antioxidants (Pihlanto, 2006).
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Milk fermentation by proteolytic lactic acid bacteria (LAB) is one of83the economical and practical methods for the production of fermented dairy products enriched 84 in bioactive peptides (Hayes et al., 2007). It has been previously reported that bovine milk 85 fermented with proteolytic strains of LAB such as Lactobacillus helveticus JCM1004 (Pan, 86 Luo, & Tanokura, 2005), Lactobacillus delbrueckii subsp. bulgaricus (Papadimitriou 87et al., 2007),
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Lactococcus lactis YT2027 and Lactobacillus acidophilus DPC6026 contain88health-beneficial bioactive peptides (Fitzgerald & Murray, 2006; Hayes, Ross, Fitzgerald, Hill, 89 & Stanton, 2006).
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Milk from different species (bovine, goat, sheep, buffalo, etc.) is used 91for production of fermented milk products (Chandan, 2004). Camel milk for fermented milk 92production
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contains well-balanced nutrients and biological components. Camel milk is 93 high in vitamin C and niacin, as well as richer in Cu and Fe than bovine milk (El-Agamy, 2006). 94 Moreover, camel milk lacks β-lactoglobulin and contains α-lactalbumin, a similar situation 95 to that in human milk. Previous research has recommended the potential use of camel96milk in the manufacture of infant formula (Salami et al., 2009). The antioxidant and ACE-inhibitory 97 property of casein-derived peptides of camel milk by digestive proteases has98already been studied (Salami et al., 2011). The significantly higher digestibility and greater 99 antioxidant
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activity of camel milk α-lactalbumin compared with that of bovine whey 100 proteins was also observed in a previous study (Salami et al., 2009). Furthermore, some reports 101 are available on peptides with a wide range of functionalities in fermented bovine milk with 102proteolytic
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strains of LAB (Fitzgerald & Murray, 2006; Hayes et al., 2007; Papadimitriou 103 et al., 2007), but there is a lack of information in the literature about biological activity104 of camel milk-
derived peptides by lactic acid fermentation. Therefore, it would be interesting 105 to investigate
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the functionalities of bioactive peptides produced during the fermentation106 of camel milk.
Based on our previous study, Lactobacillus rhamnosus PTCC 1637 107 was selected for
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this study due to its high protease activity and high mean score values for108 sensory quality in fermented bovine and camel milk (Moslehishad, Mirdamadi, Ehsani, Moosavi-Movahedi, 109 & Ezzatpanah, 2012). The objective of this study was to compare the ACE-inhibitory 110 and antioxidant activity of peptide fractions obtained from camel and bovine milk 111 fermented with
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Lb. rhamnosus PTCC 1637 during 21 days of cold storage.
Materials and methods
Milk collection
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2.1.
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117
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Bovine milk samples were obtained from a commercial dairy farm118 in Tehran province, Iran. Camel milk samples were collected from local camel producers 119 in Torkaman Sahra, Gorgan province, Iran. All milk samples were stored at 4 °C during120 transportation to the laboratory.
121 122
2.2.
Milk fermentation
123 124
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Lb. rhamnosus PTCC 1637 was obtained from Persian Type Culture 125Collection (PTCC, Tehran, Iran). It was maintained at -70 ˚C in 15% skim milk (Merck, 126 Darmstadt, Germany) plus 30% glycerol (Merck) before further experiments. The organism 127 was
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activated in sterile 10 mL aliquots of de Man Rogosa and Sharpe (MRS) broth 128 (Merck) at 37 ºC for 24 h. The cultures were centrifuged at 5000 × g for 15 min to separate 129 bacteria and
were washed twice with sterile distilled water. Biomass was then inoculated 130into skim milk
1
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12% (w/v) and incubated at 37 ºC for 24 h as pre-culture to obtain approximately 131 108 cfu mL. Fresh whole bovine and camel milk samples were pasteurised at 80 ºC for 13220 min in a
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water bath and cooled to 43 ºC. The pre-cultures were then inoculated (2%, 133v/v) into bovine and camel milk and incubated at 37 ºC for 24 h. Fermented milk samples 134 were stored at 5 ± 1 ºC for 21 days. All analyses were carried out at days 1, 7, 14 and 21 of storage. 135 The data are the mean values of two independent experiments (batches) assayed in triplicates. 136
Bacterial counts
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2.3.
137 138 139
Cell population of Lb. rhamnosus was determined by counting colony-forming 140 units
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(cfu) on MRS agar after incubation at 37 °C for 48 h in candle jar under enriched 141 CO2 (Matalon & Sandine, 1986).
142
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143
2.4.
Determination of proteolytic activity
144 145
Fermented milk (2.5 mL) was added to 5 mL of 0.75% trichloroacetic 146 acid (TCA) and
filtered through filter paper (Whatman No. 2). Proteolytic activity of filtrate 147was then measured at 340 nm in a spectrophotometer (Shimadzu, model UV-3100,148 Kyoto, Japan) as
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previously described by Church, Swaisgood, Porter, and Catignani (1983)149 and Donkor (2007).
150 151
Preparation of peptide fractions
152
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2.5.
153
The water-soluble extract (WSE) of the fermented bovine and camel 154milk was
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separated by centrifugation at 12,000 × g for 10 min at 4 ◦C and by filtration 155 through
Whatman no. 40 filter (Quiros, Hernandez-Ledesma, Ramos, Amigo, & Recio, 156 2005). The
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WSE was fractioned using ultrafiltration (UF) membranes with molecular157 mass cut off sizes of 10, 5 and 3 kDa (Amicon Ultra-15, Millipore, Carrigtwahill, Co. Cork,158 Ireland). The permeates and retentate of each stage of filtration were collected and stored 159at -20 °C until further experiments.
Measurement of ACE-inhibitory activity
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2.6.
160 161 162 163
ACE-inhibitory activity of peptide fractions of fermented milk samples 164 were
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determined by the method of Vermeirssen, Van Camp, and Verstraete (2002) 165 and Salami et al. (2011). Rabbit lung extract (Sigma-Aldrich, Munich, Germany) as the166 source of ACE was
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prepared by dissolving 1 g of rabbit lung acetone powder in 10 mL of 50 167 mM potassium phosphate buffer (pH 8.3) and 5% (v/v) glycerol. The mixture was centrifuged 168 for 40 min at 40,000 × g after overnight storage at 4 °C. The supernatant was separated169 and stored at 4 °C before analysis. An aliquot (150µL) of furanacrylolyl tripeptide (1mM) (Sigma-Aldrich) 170 was dissolved in 50 mM Tris-HCl buffer, pH 8.3, containing 400 mM NaCl, peptide 171 fractions were added to each well of ELISA plates. ACE extract was added to the mixture 172and pre-incubated at 37 °C for 2 min. The absorbance was measured at 340 nm using an ELISA 173 reader Expert
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96 (ASYS Hi-tech, Eugendorf, Austria) for periods of 25 min. The ACE activity 174 (unit L-1) of each peptide fraction was calculated according to the below formula:
175 176
(1)
177
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ACE activity = Vt. 1,000. ∆A ∆e. Vs . d
178 179
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where Vt = total volume; Vs= ACE volume; d = light path (cm); ∆e = maximum 180 change of absorbance at 340 nm; and ∆A = absorbance changes per minute. Subsequently, 181 ACE
2.7.
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inhibition was reported as IC50 (inhibitory concentration 50%) values.
Determination of antioxidant activity
182 183 184 185
Antioxidant activity of peptide fractions of fermented milk samples 186was evaluated by
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Trolox Equivalent Antioxidant Capacity (TEAC) method based on scavenging 187 of the 2,2'azino-bis-(3-ethyl-benzthiazoline-6-sulfonic acid) (ABTS; Sigma-Aldrich) 188 as described previously by Re et al. (1999) and Salami et al. (2011). ABTS assay was 189 carried out by
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oxidising ABTS (7 mM) in 2.45 mM potassium persulphate buffer for 12–16 190 h in the dark to provoke the formation of ABTS radical. ABTS•+ solution was diluted in 5191 mM phosphate
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buffer, pH 7.4, to obtain absorbance 0.7 ± 0.2 at 734 nm. ABTS•+ solution, 192 reactive towards antioxidant compounds, was added to peptide fractions. The mixture was193 incubated at 25 °C for 5 min and the absorbance was measured at 734 nm using an ELISA reader 194 Expert 96. A 6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid (Trolox) (Sigma-Aldrich) 195 standard curve was used to determine Trolox equivalent values.
196 197
2.8.
Statistical analysis
198
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199
All data were subjected to one-way analysis of variance (ANOVA) 200 using SPSS 12.0 software (SPSS INC., Chicago, IL, USA, 2002). Significant treatment means 201 were separated by Duncan’s New Multiple Range Test.
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202 203
3.
Results and Discussion
204
3.1.
Cell growth during fermentation and storage
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205 206
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207
Viable counts of Lb. rhamnosus PTCC 1637 after fermentation and 208 during cold storage time are shown in Fig. 1. The bacterial counts for fermented bovine 209and camel milk ranged initially between 8.14 to 8.86 and 8.54 to 8.90 log cfu mL-1 after fermentation, 210 respectively. In both milk types, the counts did not change significantly (P 211≥ 0.05) until day
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21 and were in the range of 8.19 to 8.40 and 8.71 to 8.80 log cfu mL-1 for212 bovine and camel cultured milk, respectively. The slow growth rate of bacterial cells during213 cold storage could be due to the preference of mesophilic to slightly thermophilic temperature 214for LAB
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(Hammes & Hertel, 2009). Moreover, the release of peptides and amino acids 215 as the nutrient supply by the proteolytic system of Lb. rhamnosus may explain the viability 216 and minimal
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decline in cell population (Pastar et al., 2003; Savijoki, Ingmer, & Varmanen, 217 2006). No significant differences (P ≥ 0.05) were found in the growth of 218 Lb. rhamnosus
between camel and bovine milk products. Contradictory results have been219 published about the growth and viability of strains used as starters in camel milk compared 220 with bovine milk. Abu-Tarboush (1996) stated that Streptococcus thermophilus and Lb. delbrueckii 221 ssp. bulgaricus grow at a faster rate in bovine milk than in camel milk. Abu-Tarboush, 222 Al-Dagal, and AL-Royl (1998) reported faster growth rate of Bifidobacterium longum 22315707 and slower
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cell growth of Bifidobacterium angulatum 27535 in camel milk than in bovine 224 milk. This probably demonstrates that the type of strain is the determinant factor that225 influences the bacterial growth rate of organisms in milk from different species.
226
3.2.
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227
Proteolysis in fermented milk
228 229
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Proteolytic activity of fermented milk was assessed during cold storage 230 based on
determination of peptide release (Fig. 2). Milk fermentation leads to formation 231 of peptides as
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the growth factor for LAB and an excess amount of peptides can be accumulated 232 in the medium (Donkor, 2007). These peptides were measured using o-phthaldialdehyde 233 (OPA) spectrophotometric assay after fermentation and during prolonged cold storage. 234 As expected, the OPA values (extent of proteolysis) increased significantly (P < 0.05) in 235both fermented bovine and camel milk until day 21. This could probably be due to the survival 236 of
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microorganisms in the presence of free NH3 groups in the medium by the237 end of storage. Furthermore, the release of cell-wall attached proteinase may occur after 238 the lysis of bacteria during shelf life (Ramchandran, 2009). The data are consistent with those239 reported by
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Nielsen, Petersen, and Dambmann (2001) and Donkor (2007). In the present 240 study, the increased proteolysis of fermented milk by Lb. rhamnosus PTCC 1637 until 241the end of
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storage, suggests that it is one of the proteolytic strains of LAB as our pervious 242 findings also revealed the same result (Moslehishad et al., 2012). Data showed that approximately 243 similar trends of proteolytic activity were obtained in fermented bovine and camel 244 milk. Our results differ from those reported by Abu-Tarboush (1996), who found higher proteolysis 245 of yoghurt starters in camel milk than in bovine milk. However, our findings might be 246due to straindependent differences.
247 248
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3.3.
ACE-inhibitory activity of peptide fractions of fermented milk
249 250
In the present study, in vitro ACE-inhibitory activity of WSE to identify 251 the biological
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activity of cultured milk samples and peptide fractions from fermented bovine 252 and camel milk separated by UF membranes with molecular mass cutoff of 10, 5 and253 3 kDa were
determined (Table 1). The ACE-inhibitory activity of milk protein-derived 254 peptides was
determined as IC50 value based on inhibiting ACE activity (Haquea et al. 255 2009). As shown in
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Table 1, the lowest IC50 value was 1.45±0.02 mg mL-1. The IC50 value was 256 considerably
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lower than that reported by Qian et al. (2011) for the most potent ACE-inhibitory 257 peptide fraction obtained from fermented bovine milk by Lb. delbrueckii ssp. bulgaricus 258 LB with an IC50 value of 67.71±7.62 mg mL-1. This may suggest that ACE-inhibitory259 peptide formation is related to the bacterial strains and culture conditions used in the fermentation 260 process. Furthermore, Ferreira et al. (2007) reported that amino acid residues/sequences 261
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greatly contribute to ACE-inhibitory potency of bioactive peptides. Therefore, 262 the differences in the primary structure of camel milk proteins than in bovine milk (Kappeler, 263 Farah, & Puhan, 1998) may also influence the ACE-inhibitory activity of camel milk 264peptide fractions.
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Table 1 also shows that in the case of both bovine and camel milk,265 the lowest IC50 values were observed in the <5 kDa peptide fractions obtained after hydrolysis 266 of milk
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proteins with proteolytic enzymes of Lb. rhamnosus. Generally, most of the 267demonstrated antihypertensive peptides are short peptides with a Pro residue at the C-terminus 268 (Mizuno, Nishimura, Matsuura, Gotou, & Yamamoto, 2004). Short peptides containing 269 Pro at the Cterminal end are resistant against digestive enzymes and more readily transfer 270 to the blood stream (Korhonen & Pihlanto, 2006). Gobbetti, Ferranti, Smacchi, Goffeedi, 271 and Addeo (2000) reported the formation of ACE-inhibitory peptides released from milk 272 proteins by starter cultures containing proline-specific peptidases during fermentation. 273 Lb. rhamnosus
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possesses a proteolytic system that includes a proline-specific peptidase like 274 proline-specific aminopeptidase (PepR) and X-prolyl-dipeptidyl aminopeptidase (PepX; Pastar 275 et al., 2003; Savijoki et al., 2006) that subsequently may result in accumulation of bioactive 276 ACE-
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inhibitors in fermented milk. This may explain the results obtained in this277 work which indicates that Lb. rhamnosus PTCC 1637 is capable of producing potent ACE-inhibitory 278
peptides from bovine and camel milk proteins, especially from caseins containing 279 a high level
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of proline residues. These results are in agreement with those obtained by280 Hernández-
Ledesma, Amigo, Ramos, and Recio (2004) when studying ACE-inhibitory 281activity of bovine
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milk fermented with Lb. rhamnosus CECT 287T. They also reported the release 282 of ACEinhibitory activity peptides during fermentation of bovine milk.
283
The ACE-inhibitory values of WSE found for cultured bovine milk 284 were in the range of 2.483±0.148 to 3.947±0.029 mg mL-1 and 2.223±0.052 to 3.930±0.118285 mg mL-1 for camel milk during 21 days of storage. The results indicated that the IC50 values,286 especially in camel
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milk, were considerably lower than that reported by Chen et al. (2010) for287 koumiss whey (52.47±2.87 mg mL-1). Moreover, in this study higher ACE-inhibitory activity 288 was observed from cultured camel milk peptide fractions than bovine milk counterparts.289 This may be
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explained by the presence of higher proline content in the primary structure 290of camel milk caseins than in bovine milk (El-Agamy, 2006). Our results were in agreement 291 with those
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reported by Salami et al. (2011) that showed a significant antihypertensive 292 property of camel milk casein following enzymatic digestion.
293
The extent of ACE inhibition did not follow any specific trend during 294 21 days of
storage at 5 °C (Table 1). This finding suggests that some of the peptides 295 produced at the early or intermediate stage of hydrolysis may degrade and new ones may 296 be produced due to the secondary proteolysis throughout the shelf life in both fermented milk297 samples. Similar trends were previously reported by Donkor (2007). In contrast, Nielsen, Martinussen, 298
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Flambard, Sørensen, and Otteet (2009) found that the ACE-inhibitory activity 299 of fermented milk using Lc. lactis and Lb. helveticus strains 1198 and 1263 increased during 300 7 days of cold storage. Consequently, the different results on the trends of ACE-inhibition 301during storage
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period may be explained by the significant effect of bacterial strains on hypotensive 302 peptide production during refrigerated storage time in fermented milk products. 303 304
3.4.
Determination of antioxidant activity
305
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306
The antioxidant activity of peptide fractions (5-10, 3-5, and < 3 kDa) 307 and WSE of
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fermented bovine and camel milk by Lb. rhamnosus showed that fermentation 308 increased the ABTS radical scavenging activity (Figs 3 and 4). This may be due to the hydrolysis 309 of αS1and β-casein by proteolytic/peptidolytic enzymes of Lb. rhamnosus (Varmanen, 310 Rantanen, Palva, & Tynkkynen, 1998; Pastar et al., 2003). Moreover, production of 311 PrtR proteinase,
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PepO endopeptidase (NH2–Xn↓Xn–COOH), PepR (NH2–Pro↓X–COOH)312 and PepX (NH2– X–Pro↓Xn–COOH) by Lb. rhamnosus has been previously reported by Pastar 313 et al. (2003) and Savijoki et al. (2006).
314
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The 5-10 kDa peptide fractions exhibit the highest radical scavenging 315 activities compared with lower molecular masses of peptides in both fermented milk. 316The level of
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TEAC values of 5-10 kDa peptides were in the range of 110.41 to 745.35317 µM and 844.08 to 1737.88 µM in fermented bovine and camel milk, respectively (Figs. 3 and 318 4). Peptide fractions extracted from camel milk fermented by Lb. rhamnosus showed319 higher antioxidant activity than in bovine milk. The findings suggest that not only peptide size, 320but also the nature and composition of peptides, which is not similar in fermented bovine 321 and camel milk, plays an important role in quenching of ABTS radicals and antioxidant efficiency. 322 The presence of hydrophobic and aromatic amino acid residues in323 peptides is a determinant factor in radical scavenging (Ajibola, Fashakin, Fagbemi, & 324 Aluko, 2011). 13
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Certain amino acids such as His (imidazole group), Trp (indolic group), Tyr 325(phenolic group), Phe (aromatic amino acid) and Pro (hydroxyl radical scavenger) correlated 326 with antioxidant capacity. Furthermore, Met and Cys are effective in quenching of free radicals 327 due to their
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ability to donate sulphur hydrogen (Ajibola et al., 2011; Xiong, 2010). It was 328 hypothesised that the higher level of Pro, Met, Ile, Trp residues in camel caseins than in329 bovine milk leads to an increase in free-radical scavenging activity of peptide fractions extracted 330 from
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fermented camel milk compared with bovine milk samples (El-Agamy, 2006). 331
In the current study, the WSE of both fermented milk products showed 332 significantly
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(P < 0.05) the highest antioxidant activity compared with peptide fractions 333 (Figs 3 and 4). The TEAC values ranged from 277.35 to 930.41 µM and 1417.88 to 2035.61 334 µM in fermented bovine and camel milk, respectively. This may be due to the synergistic effect 335 between peptides and milk proteins enhancing antioxidant capacity (Kwak, Seo, &336 Lee, 2009). Radical scavenging capacity of milk proteins has been previously reported 337 by Chiang and
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Chang (2005). Furthermore, Salami et al. (2010) have reported higher antioxidative 338 activity of camel whey proteins than bovine whey, which is in accordance with our 339 results, where more pronounced ABTS radical scavenging activity of WSE of fermented340 camel milk by Lb. rhamnosus than bovine milk was observed.
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341
As shown in Fig.3, the highest TEAC value (2053.61 µM) was observed 342 in the WSE
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of fermented camel milk by Lb. rhamnosus on day 21. This result is considerably 343 higher than that reported previously by Virtanen, Pihlanto, Akkanen, and Korhonen (2007) 344 for Leuconostoc mesenteroides ssp.cremoris (700 µM) and Lactobacillus jensenii 345 (610 µM). This may suggest that the development of antioxidative activity during milk fermentation 346 is a strain-dependent factor. Compared with protease digestion by trypsin and347 papain, as previously reported by Pattorn & Hongsrragas (2012) and Zhao et al. (2010) 348 respectively, milk fermentation using Lb. rhamnosus, especially in camel milk, revealed 349 higher scavenging
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activity. The results may suggest the potential use of fermentation in enhancing 350 natural antioxidant factors of bovine and camel milk products.
351
In the present study, the antioxidant capacity of fermented bovine352 and camel milk
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products did not decline at the end of the storage period. Noticeably, the extent 353 of TEAC values in both bovine and camel milk after fermentation and at weekly intervals 354 was timedependent and the highest value (P < 0.05) was observed on day 21 (Figs355 3 and 4). This
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finding may be due to the presence of vital microorganisms and the release 356of enzymes from them within the food matrix during storage time. The data obtained in this357 study showed the
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increased proteolysis and survival of Lb. rhamnosus during cold storage which 358 may result in high level of antioxidant activity at the end of the storage period. Our finding 359 was also in agreement with those reported by Virtanen et al. (2007) which revealed the 360relationship between the development of antioxidant activity and a high degree of proteolysis. 361
Conclusion
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4.
362 363 364
Milk fermentation resulted in the release of peptides as the growth365 factor for Lb.
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rhamnosus. Fermentation of milk by Lb. rhamnosus as a proteolytic strain366 can cause to release ACE-inhibitory and antioxidant peptides from bovine and camel milk 367 proteins. This
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work also confirms that the enhancement of proteolytic activity contributed 368to the increased antioxidant activity of both fermented milk types during cold storage. ACE-inhibitory 369 and antioxidant activity of fermented camel milk by Lb. rhamnosus were more 370 pronounced than bovine milk. A potential for using these findings is the development of fermented 371 camel milk by LAB as a novel food product containing ACE-inhibitory and antioxidant 372peptides. 373
Acknowledgements
374
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375
The support of the University of Tehran, Iranian Research Organization 376 for Science and Technology (IROST), Iran National Science Foundation (INSF), Iran377 Dairy Industries
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Company (Pegah) and Islamic Azad University, Science and Research Branch 378 of Tehran are gratefully acknowledged.
379 380
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Table 1
1
PTCC 1637 during cold storage. a
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ACE inhibitory activities (IC50) of peptide fractions and water-soluble extract2 of fermented camel and bovine milk by Lactobacillus rhamnosus 3 4
Storage Fraction size (kDa) time (d) 5-10 3-5
Water soluble extract
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Fermented milk
<3
1
1.790±0.010 aA
2.545±0.185 bA
1.830±0.160 acA
3.930±0.118dA
Bovine
1
2.490±0.040 aB
3.810±0.175 bB
1.510±0.030 cA
3.947±0.029 bA
Camel
7
4.475±0.045 aA
1.450±0.015 bA
2.960±0.040 cA
2.423±0.200 dA
Bovine
7
4.200±0.006 aA
2.040±0.060 bB
3.840±0.073cB
3.653±0.045 cB
Camel
14
3.405±0.205 aA
3.500±0.050 aA
2.250±0.077 bA
2.873±0.094 cA
Bovine
14
4.325±0.005 aA
1.510±0. 175bB
2.183±0.024 cA
2.483±0.148 cA
Camel
21
2.210±0.064 aA
2.310±0.036 aA
3.710±0.020 bA
2.223±0.052 aA
Bovine
21
2.830±0.082 aB
1.803±0.095 cB
3.223±0.187 aB
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4.095±0.195 bB
5
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IC50 is defined as the concentration (mg mL-1) required to inhibit 50% of ACE 6 activity. Data are means ± standard error of the mean of triplicate measurements of two independent experiments (batches); values with7 different superscript lower case letters in a row and with different superscript upper case letters in a column are statistically significant at P < 0.05. 8 9
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Camel
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Cell population (log cfu mL-1)
a/A a/A
a/A
a/A
a/A
a/A
a/A
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a/B
Fermented bovine milk
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Fermented camel milk
1
2 3 4
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1 2 3
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4
d
c
bc
a
b
b
a
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c
b
1
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1
c/C
c/B
TEAC (µM)
d/A
a/A
a/AB a/C
a/C
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c/B
b/C
a/B
b/D
b/A
b/C c/A
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b/B b/B
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2 3
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TEAC (µM)
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1 2
c/C
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a/B
c/B b/AB
b/C b/C
a/A a/A a/A
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c/A
a/A
a/A
a/A
b/B
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b/B b/B
3 4
5
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Figure legends
1 2
Fig. 1. Population of bacteria in fermented bovine and camel milks 3with Lactobacillus ), 7 (
), 14 ( ),421( ). Error bars
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rhamnosus PTCC 1637 during cold storage at days 1(
show standard error of the mean of triplicate measurements of two independent 5 experiments (batches). Mean values with different small letters for the same fermented6 milk type and in
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capital letters for the same storage time are significantly different (P < 0.05). 7 8
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Fig. 2. Proteolytic activity of fermented bovine () and camel () milks 9with Lactobacillus rhamnosus PTCC 1637 during cold storage. Error bars show standard error 10 of the mean of triplicate measurements of two independent experiments (batches). Different 11 letters indicate
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significant differences in fermented milk during cold storage (P < 0.05).
Fig. 3. Antioxidant activities of peptide fractions 5-10 kDa (
12 13
), 3-5 kDa (14 ), <3 kDa (
) and
water-soluble extract (WSE) ( ) of fermented camel milk with Lactobacillus 15 rhamnosus PTCC
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1637 during storage. The data measured as Trolox equivalent antioxidant16 capacity (µ M). Error bars show standard error of the mean of triplicate measurements 17 of two independent
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experiments. Mean values with different small letters for the same storage 18 time and in capital letters for the same molecular mass peptide fraction or WSE during cold storage 19 are significantly different (P < 0.05).
20 21
Fig. 4. Antioxidant activities of peptide fractions 5-10 kDa ( water-soluble extract (WSE) (
), 3-5 kDa (22 ), <3 kDa (
) and
) of fermented bovine milk with Lactobacillus 23 rhamnosus PTCC
1637 during cold storage. The data measured as Trolox equivalent antioxidant 24 capacity (µ M).
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Error bars show standard error of the mean of triplicate measurements25of two independent experiments (batches). Mean values with different small letters for the same 26 storage time and in capital letters for the same molecular mass peptide fraction or WSE during 27 cold storage are significantly different (P < 0.05).
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