Introduction

Milk derived bioactive peptides are considered as prominent ingredients for various health promoting functional foods targeted for heart, bone and digestive system as well as improving immune defense, mood and stress control. The functional milk products with these bioactive peptides can be produced by enzymatic hydrolysis of milk with proteolytic enzyme or by fermentation of milk with lactic acid bacteria (LAB). Now a days, the consumers pay lot of attention towards the food security and functionality due to the relation between food and health. Hypertension is a risk factor for coronary heart disease (Collins et al. 1990). Angiotensin-I converting enzyme (ACE; kininase II; EC 3.4.15.1) is a Zn-metallopeptidase and plays an important role in regulating blood pressure located in different tissues which plays a key physiological role in the regulation of local levels of several endogenous bioactive peptides (Bruneval et al. 1986; Ondetti and Cushman 1982). ACE has been classically associated with the renin-angiotensin system which regulates peripheral blood pressure, where it catalyzes both the production of the vasoconstrictor angiotensin-II and the inactivation of the vasodilator bradykinin. ACE inhibition results mainly in an antihypertensive effect but may also influence different regulatory systems involved in modulating blood pressure, immune defense and nervous system activity (Meisel 1993). Naturally occurring peptides in snake venom were the first reported competitive inhibitors of ACE (Ferreira et al. 1970; Ondetti et al. 1971). Thereafter, many other ACE inhibitors were discovered from enzymatic hydrolysates or the related synthetic peptides of bovine and human caseins (CNs), as well as plant and other food proteins (Smacchi and Gobbetti 1998).

Recently, researchers have reported that milk proteins contain the ACE-inhibitory peptide sequence, which can be released by proteolysis during milk fermentation by some strains of LAB (Yamamoto et al. 1994a, 1999). Takano (1998) reported that peptides derived from Calpis sour milk (a Japanese soft drink fermented by L. helveticus and Saccharomyces cerevisiae) reduced blood pressure. Milk protein derived ACE-inhibitory peptides are inactive within parent protein and are released and activated by enzymatic proteolysis of lactic-acid bacteria. Among lactic-acid bacteria, L. helveticus has been shown to exhibit strong proteolytic activity in milk-based media. The proteolytic systems of L. helveticus are composed of a cell-envelope proteinase and more than 10 intracellular peptidases, including endopeptidases, aminopeptidases and X-prolyldipeptidyl aminopeptidase (Exterkate 1995). The cell-envelope proteinase is a key enzyme in the proteolytic system. It catalyzes the initial steps in the degradation of protein into different oligopeptides. The degradation products are transported across the cell membrane by 10 different amino acid transport systems, including an oligopeptide (Opp), dipeptide (DtpP) and tripeptide (DtpT) transporters (Law and Haandrikman 1997). Then the peptides are further hydrolysed into amino acids or small peptides by intracellular peptidases.

The proteolytic system of LAB can contribute to the liberation of health enhancing bioactive peptides from milk. The latter may improve absorption in the intestinal tract, stimulate the immune system, exert antihypertensive or antithrombotic effects and antioxidative activity or function as carriers for minerals especially calcium.

In this study, total 12 lactic acid bacterial (LAB) strains were screened and selected. Lactobacillus acidophilus used as an adjunct culture with standard dahi culture NCDC-167 to produce fermented milk containing ACE-inhibitory peptides. The incubation period and pre heat treatment to milk before incubation (the time–temperature combination) was optimized using RSM. The ACE-inhibitory peptides were purified, isolated, sequenced and their bioactivity was characterized.

Materials and Methods

Milk and Cultures

The buffalo milk was procured from cattle yard, NDRI Karnal and standard mixed dahi culture of Streptococcus thermophilus and Lactococcus lactis NCDC-167(BD4) with L. acidophilus NCDC-15 were procured from the center of National Collection of Dairy Culture (NCDC), NDRI Karnal.

Heat Treatment of Milk

For the development of final products, pre heating of milk was finalized for the combination of temperature and time by using response surface method (RSM). For this purpose, the preliminary trials were conducted to investigate the effect of temperature (60–90 °C) and time from 2 to 12 h on ACE inhibitory activity in lassi.

Propagation and Maintenance of Lactobacillus Cultures

All the Lactobacillus cultures were propagated in 10 ml sterile de Man-Rogosa-Sharpe (MRS) broth and maintained in litmus milk at refrigerator until use. These were periodically sub-cultured in the same medium once in a week. Each culture was activated by sub-culturing before use and purity was always ascertained by Gram’s staining. One set of cultures was stored at −80 °C in MRS broth containing 20 % glycerol as a stock.

Preparation of Cell Free Supernatant

The fresh Lactobacillus cultures were inoculated into skim milk @ 2 percent and incubated at 37 °C for 8, 10 and 12 h. After incubation, the supernatants of fermented skim milk was obtained by adjusting the pH to 4.6 and centrifugation at 10,000 rpm for 10 min at 4 °C (Kubota centrifuge, Tokyo, Japan). The supernatant was collected, filtered through sterilized (0.22 μm membrane filter, Millipore). This supernatant was used for assessing ACE inhibitory activity and protein content by Lowry’s (1951) Method.

Angiotensin Converting Enzyme (ACE) Inhibition Properties

The assay for the Angiotensin Converting Enzyme (ACE) inhibitory activity based on the liberation of hippuric acid from hippuryl-l-histidyl-l-leucine (HHL) catalyzed by ACE was measured by the method of Cushman and Cheung (1971) as modified by Ledesma et al. (2005). The hippuric acid liberated by the ACE was extracted with 1.5 ml ethyl acetate by centrifugation at 3000g for 10 min, then heat evaporated at 95 °C for 10 min, redissolved in 1 ml distilled water and measured spectrophotometrically at optical density of 228 nm. The extent of inhibition was calculated as follows:

$${\text{Percent inhibition}} = \left( {{\text{B}} - {\text{A}}} \right)/\left( {{\text{B}} - {\text{C}}} \right) \times 100$$

where A = absorbance in the presence of ACE and ACE inhibitory component, B = absorbance without ACE inhibitory component, and C = absorbance without ACE.

Inhibition was expressed as the concentration of component that inhibits 50 % of ACE activity (IC50), and 1 unit of ACE inhibitory activity was expressed as the potency showing 50 %.

Purification of Peptides Solvent

The supernatants of lassi samples were purified and concentrated for bioactive peptides by using the Solid Phase extraction (SPE) tubes (Discovery SPE tubes, Sigma-Aldrich Co.) A column of (2.0 × 30 cm) SP Sephadex C-25 (Sigma) was packed and equilibrated with Solvent A [Water and TFA (1000:1, v/v) (HPLC grade)] and sample was loaded on to the column at the flow rate of 0.5 mL per min. Then the column was washed with 70:30 (Acetonitrile: Water HPLC grade) by 2 column volume. Finally, the column was washed (3–4 column volume) with Solvent B [Acetonitrile and TFA (1000:0.8, v/v)] and eluate was collected and lyophilized. Each time the column was equilibrated before loading the sample, after previous washing, for the better contact between samples and hydrophobic solid phase.

Separation and Identification of Peptide Peak

Separation and Identification of peptide peaks was done by following the procedure of Hernandez et al. (2005b). The reverse phase HPLC system (Dionex, Ultimate 3000, USA) was used for profiling of the peptides. The concentrated supernatant fraction of solid phase extraction was diluted with freshly prepared 0.05 percent trifluoroacetic acid (TFA). The acidic mixture was centrifuged for 20 min at 5000 rpm. The supernatant, containing TFA soluble peptides were filtered (0.22 μm). The separation was performed using C18 column (AG 120 A, 5 μm with conditions: Column stationary phase: Dionex C18, 5 μm, 120 A°, 250 × 4.6 mm ID; Reverse phase; UV detector wavelength: 214 nm; Solvent A: Water and TFA (1000: 1, v/v) (HPLC grade); Solvent B: Acetonitrile and TFA (1000:0.8, v/v) (HPLC grade); Flow rate: 0.8 mL/min; Run time: 140 min; Maximum pressure: 400 kgmf; Injection volume: 20 μl and Binary Gradient Programming.

Separation and Collection of Peptides Fraction by FPLC

Separation and Collection of peptides Fraction was done by using AKTA purifier (GE Healthcare Biosciences, Hong Kong) equipped with RESOURCE RPC 3 mL (Reverse phase column) by equilibration of the column with 10 column volumes of Solvent A. The freeze dried peptide sample was dissolved in solvent A and filtered through 0.22 syringe filter, loaded to 0.5 mL loop fitted to the equipment and separation of the peptide was carried out using binary gradient. The several runs were performed and fractions were collected i.e. fractionI (17–50 min.), fractionII (55–90 min.) and fractionIII (95–110 min.) individually. The dried fractions were dissolved in 100 μl distilled water and assessed for its ACE inhibitory activity and protein content.

Peptide Identification

The peptide samples were sequenced at Proteomics International Pvt Ltd. Australia through Techno Concept, New Delhi. In order to identify the bio-functional peptides, the samples were analysed by electrospray ionization mass spectrometry using the Ultimate 3000 nano HPLC system [Dionex] coupled to a 4000 Q TRAP mass spectrometer [Applied Biosystems]. The peptides were loaded onto a C18 PepMap100, 3 mm [LC Packings] and separated with a linear gradient of water/acetonitrile/0.1 % formic acid (v/v). Spectra were analysed to identify proteins of interest using Mascot sequence matching software [Matrix Science] with taxonomy set to other mammalian.

Results and Discussion

Standardization of Incubation Time and Preheat Treatment

The culture of lactobacillus i.e. L. acidophilus (NCDC-15) for ACE inhibitory activity was further studied to standardize the incubation time for the optimum ACE inhibitory activity at 37 °C for 8, 10 and 12 h.

The significant difference at 5 % level was recorded for ACE Inhibitory activities i.e. 27.22 ± 0.048, 26.62 ± 0.051 and 32.79 ± 0.058 (IC50 μg/ml) in the cell free extracts of fermented skim milk after 8, 10 and 12 h incubation, respectively (Table 1). The minimum IC50 value was observed at 10 h time of incubation and finalized for the further study.

Table 1 Standardization of incubation time for the fermentation of skim milk with L. acidophilus @ 2 % at 37 °C with report to ACE inhibitory activity
Full size table

The preheating treatment of milk affected the ACE Inhibitory activity of fermented milk due to the partial heat denaturation of milk protein resulted into easy asses to enzymatic hydrolysis, rendering milk as a more stimulatory growth medium for starter cultures, reduction of the redox potential and elimination of growth inhibitors which improved the physical properties of fermented milk and prevention of hydrolytic rancidity through inactivation of lipases (Kessler 1981; Krishna and Shankar 1986). The biological activities and physical properties are directly related to the production of bioactive peptides during the fermentation of milk. Further, the bioactive peptides functionality depends on the proteolytic system of lactic acid bacteria used in the fermentation. During short term fermentation cell envelope protease (CEP) are secreted which causes the hydrolysis of milk protein and releases bioactive peptides (Mierau et al. 1997; Juillard et al. 1995). The action of various proteases on the milk protein is enhanced by the preheat treatment which lead to partial denaturation of the whey proteins and antihypertensive property (Elizabete et al. 2007; Costa et al. 2007). Gul and Sibel (2009) studied the effect of heat treatment on the angiotensin converting enzyme activity of some legume species. They reported that the 50 min heat treatment enhanced the release of bioactive peptides. Moreover, during the preparation of ‘lassi’ traditionally in villages the milk is heated at higher temperature for longer time (i.e. 70–90 °C for 1–10 h). So there was need to optimize the intensity of preheat treatment for the preparation of bio-functional ‘lassi’ with substantially biological activities along with the required organoleptic properties (similar to that of the traditional lassi) and was done by using response surface methodology (RSM) with levels coded as −1, 0, +1. The ACE inhibitory activities varied from 29.90 ± 0.004 to 76.43 ± 0.012 μg protein/ml (Fig. 1). Initially, ACE inhibitory activity found decreased from 4 to 8 h at temperature between 60 and 80 °C showed increased in the IC50 value. But after 8 h, IC50 value decreased means ACE inhibitory activity increased for same temperature range i.e. 60–80 °C. Further on heating from 80 to 90 °C the IC50 value again increased with increase in heating time. The minimum IC50 value for the ACE inhibitory activity is indicated between temperature 75–82 °C (Fig. 1).

Fig. 1
figure 1

Response surface plot for the effects of preheating temperature on ACE-inhibitory activity of supernatants of skim milk prepared from L. acidophilus

Full size image

The statistically significant model for ACE inhibitory activity (p value <0.5) from Table 2 indicates that the model is suitable to predict the ACE inhibitory activity with respect to preheating time and temperature. The experimental values for ACE inhibitory activity was 36.62 ± 0.051 mg of protein/ml i.e. 27.5 % lower (high ACE inhibitory activity) than predicted value.

Table 2 ACE-inhibitory activities of fermented skim milk at optimum time and temperature of preheating selected by response surface methodology (RSM)
Full size table

Separation and Identification of Bioactive Peptides

The lassi was subjected to centrifugation at 1000 rpm for 10 min after adjusting the pH at 4.6 and supernatants were collected and passed through the 0.22 μm sterile syringe filter membrane to remove the bacterial cell. Then the filtrate was subjected to solid phase extraction using acetonitrile. The elute from solid phase extraction was concentrated and applied to RP-HPLC at C18 (ODS) column and eluted with gradient of solvent-A (water and TFA) and solvent-B (Acetonitrile and TFA). The RP-HPLC chromatograms were presented in Fig. 2. The whole chromatograph divided into three fraction i.e. fraction-I (17–50 min.), fraction-II (55–90 min.) and fraction-III (95–110 min.). All three fractions were collected, and an ACE inhibitory activity was determined.

Fig. 2
figure 2

RP-HPLC profiles of supernantants of experimental lassi (L1) prepared by using L. acidophilus (NCDC-15) as an adjunct culture with standard dahi culture (NCDC-167)

Full size image

Similarly the ACE inhibitory activities of all three fractions were presented in the Fig. 3. The fraction-III showed minimum IC50 value i.e. 14.57 ± 0.72 µg/ml which means highest ACE inhibitory activity when compared with fraction-I and fraction-II. The ACE inhibitory activity of the fraction-II is more than the fraction-I. Similarly, Donkor et al. (2007) also reported that the different HPLC fraction of water soluble extracts of yoghurt showed different ACE inhibitory activity.

Fig. 3
figure 3

ACE-inhibitory activity (IC50 value) of FPLC Fractions and supernatants of experimental lassi(L1) prepared by using L. acidophilus (NCDC-15) as an adjunct culture with standard dahi culture (NCDC-167)

Full size image

The values are mean ± SD, CD at 5 % = 3.21 (n = 3) and IC50 = value of protein content needed to inhibit 50 % of ACE-inhibitors. Values with different small letters superscripts differ significantly at P ≤ 0.05.

Identification Peptides from Different FPLC Fraction of Lassi

Total 45 peptides have been identified from the pooled FPLC fraction-II and III (Fig. 3) and presented in the Table 3. Out of these 30 peptides (as whole or a part of sequences) are well known ACE inhibitory peptides and reported by a number of workers mentioned in Table 3 (Contreras et al. 2009; Gobbetti et al. 2000; Quiro’s et al. 2007; Nakamura et al. 1995; Maeno et al. 1996; Miguel et al. 2006; Perpetuo et al. 2003; Pihlanto et al. 1998; Yamamoto et al. 1994b; Smacchi and Gobbetti 1998). Other 15 peptides have shown antioxidant, opioid, cytomodulatory, immunomodulating and antimicrobial activities as reported by the various workers (Table 3). The two peptides KVLPVPQK (β-CN f169–176) and YQEPVLGPVRGPFPIIV (β-CN f193–209) have the same sequence as reported by Maeno et al. (1996) and Yamamoto et al. (1994b), respectively as ACE inhibitory peptides.

Table 3 Peptides identified in Fermented milk (lassi) prepared by using L. acidophilus (NCDC-15) as an adjunct culture with standard dahi culture of S. thermophilus and L. lactis (NCDC167)
Full size table

The structural functional relationship of ACE inhibitory peptides indicates that their C-terminal tripeptide residues play a predominant role in competitive binding to the active site of ACE. Among the most favorable C-terminal amino acid are aromatic amino acids and the imino acid proline, but the peptide which have terminal dicarboxylic amino acid binds weakly with ACE (Gomez et al. 2002). Further Lopez-Fandino et al. (2006) also reported that the binding ACE is strongly influenced by the C-terminal sequence, whereby hydrophobic amino acid e.g. Proline are more active if present at each of the three C-terminal positions. In addition the presence of the positive charge of lysine and arginine as the C-terminal residue may contribute to the inhibitory potency. Similarly Pihlanto et al. (2010) made a statement that certain structural characteristics are evident of candidate peptides that exert an antihypertensive effect, for instance, proline, tyrosine or tryptophan most commonly appear in the carboxyl terminal.

Most of the identified peptides in fraction-II and III as enlisted in Table 3 match the full and some partial structural characteristics as described by the above mentioned researchers. The total 45 identified peptides (Table 3) from fermented milk (lassi) out of which 27, 10, 4 and 1 number of peptides showed the presence of proline, lysine, tryptophan and tyrosine in one of the three C-terminal amino acid residue, respectively. While proline in 4 peptides, lysine in 6 peptides and tryptophan in 4 peptides have been detected at C-terminal position.

Conclusion

The Lactobacillus acidophillus NCDC-15 showed minimum IC50 value for ACE-inhibitory activity (36.62 ± 0.051 mg of protein/ml). The ACE Inhibitory activities was increased by giving preheat treatment to milk before culturing. Total 45 peptides have been identified from the lassi prepared from L. acidophilus as an adjunct culture with standard mixed dahi culture of S. thermophilus and L. lactis. All the peptides identified are the fragments of β casein i.e. β-CN f47–56, β-CN f47–57, β-CN f199–209, β-CN f176–182, β-CN f176–183, β-CN f176–184, β-CN f1–7, β-CN f57–68, β-CN f166–175, β-CN f195–206, β-CN f195–207, β-CN f195–209, β-CN f94–106 and β-CN f169–176. The bioactive peptide found in lassi showed partially or completely homology to that the milk protein bioactive peptides having ACE inhibitory, immunomodulatory, antioxidant, opioid and cytomodulatory activites. The results suggest that the bioactive peptides in fermented milk products can be increased using controlled fermentation and proteolytic starter strain. Fermentation is easy and cost effective method to generate the bioactive peptides in fermented milk products.