People for an activity of lactic acid bacteria,

aware of their health and have taken more attention toward nutritional values
and functional properties of food, which present in dairy products. Fermented
milk products are becoming increasingly popular because of the numerous health
benefits associated with highly digestible nutrient especially with lactose
maldigestion and high bioactive peptides 1, 2, 3. So many peptides with
bioactive characteristic have been isolated from fermented dairy products
including fermented milk, chess and yogurt. These peptides have many benefits
in inhibition many diseases like as cancer and hypertension.

       Anti-hypertension properties of peptides
returned to the mechanism of action of Angiotensin Converting Enzyme (ACE).
Angiotensin Converting Enzyme converts angiotensin I to angiotensin II, which
causes vasoconstriction and stimulating the secretion of aldosterone by the
adrenal glands 4, 5, 6. Inhibition of angiotensin I-converting enzyme (ACE)
is considered as a useful therapeutic approach in the treatment of high blood
pressure in both diabetic and nondiabetic patients 4, 7. Most of the
milk-derived peptides have multiple functions are involved in the production of
bioactive substances 8, 5. Proteolysis leads to the production of bioactive
peptides which have positive effects on human health. On the other hand for an
activity of lactic acid bacteria, proteolysis is the most important biochemical
process occurring in sour milk products during fermentation and storage 9.
Improvements of ACE inhibitory activity during fermentation depend on the
degree of hydrolysis 10. Lactic acid bacteria (LAB) are known specific in the
production of ACE inhibitors during fermentation. 11, 12.

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      Sometimes a number of inhibitors are more
efficient with the enzymes such as trypsin, pepsin and carboxypeptidase A. Some
of these inhibitors show greater efficiency in vivo after the effect of
digestive enzymes on casein like dipeptide tyrosine – proline. Yogurt-like
products contain two tripeptides IPP (Isoleucine-Proline-Proline) and VPP
(Valine-Proline-Proline) which are resistant to gastric digestion while others
decrease in performance 13. There are numerous data in this field, including
consumption of products containing inhibitors; particularly VPP and IPP reduce both
systolic and diastolic blood pressure 14, 15. However, inhibitors in food are
not powerful as drugs used to treat high blood pressure, but also they have
mild activities that can be viewed as a natural functional food in the diet and
placed in the daily diet. Some oral ACE inhibitors are currently used as
clinical antihypertensive drugs such as captopril, enalapril, and lisinopril
16. Enalaprilat is the active antihypertensive drug, which is dosed as the
prodrug, enalapril. By chemical hydrolysis from the prodrug enalapril, the
absorption after oral intake will be improved 17.

The aims of this study were to evaluate effects of sodium caseinate, M.piperita
extract and storage time on the viability of L. casei, ACE-inhibitory
activity and proteolysis. In addition, this study can be a starting point for
practical use of peppermint extract in dairy products in near future. As well,
it can be the effect on improving food health and safety in the society.

Materials and Methods


     Skim milk powder (SMP) was
purchased from the Kaleh Dairy Company (Urmia, Iran). DVS (Direct Vat Set)
freeze-dried yogurt culture (YC-350: mixed cultures of L. delbrueckii
subsp. Bulgaricus and S. thermophilus) was purchased from Chris
Hansen, Denmark. L. casei LAFTI® L26 was obtained from DSM Food
Specialties (Moorebank, NSW, Australia). Sodium caseinate with 78.95 % protein
(Milad Khorasan, Iran), with water and alcoholic extract of peppermint
(Zardband Iran), were used. Lyophilized powder of rabbit lung
angiotensin-converting enzyme, enalapril, O-phthaldialdehyde and the substrate
of ACE, FAPGG (N-3-(2-Furyl) acryloyl-L-phenylalanyl-glycyl-glycine) were
purchased from Sigma–Aldrich (St. Louis, MO).

Experimental design and statistical analysis

      The face-centred central composite design
was used and the results were submitted to the ANOVA using SAS (version 9.1,
SAS Institute Inc.) and the plots were drawn by Matlab software (version
7.10.0449, The MathWorks Inc.). The response surface methodology (RSM) is a
collection of mathematical and statistical techniques for designing
experiments, modeling, investigating the effect of some factors on one or more
dependent variables 18. The independent variables were sodium caseinate,
peppermint extract and storage time. Actual and coded values of the factors are
given in Table 1. Twenty yogurt samples were evaluated according to this table
design with three variables and three levels for each variable. Data were fitted
to the second-order polynomial.


Where Y represented predicted response; ?0, the constant
term; ?i, linear coefficient; ?ii, squared coefficient and ?ij, interaction
coefficient and X were the independent coded variables.


Yogurt preparation

Low-fat yogurt was prepared by using skimmed
milk (Kaleh, Urmia, Iran) that was standardized in 12, 14 and 16 g /100 ml
total solids with skim milk powder and sodium caseinate (0-4%). According to
the Table 1. sodium caseinate and peppermint extract were added into pre-warmed
(41?C) pasteurized nonfat milk. DVS freeze-dried yogurt culture (YC-350: mixed
cultures of L. delbrueckii subsp. Bulgaricus and S.
thermophilus) was used at 1% (w/v) and L. casei as the probiotic
culture was applied at 10 g/100 kg of milk19 then aliquoted (250 ml) into
disposable plastic containers. Plain-yogurt was prepared essentially in the
same manner. Yogurts were incubated (43±1 ?C) until the pH was reduced to 4.5
followed by refrigeration (5±1 ?C) up to 20 days



of pH and TA (titratable acid)

pH was determined after calibrating digital pH
meter by 4 and 7 standard buffers. The electrode of pH meter was set directly
in yogurt samples and then pH was recorded. Acidity was determined by titrating
with NaOH 0.1 N in the presence of 2 drops of phenolphthalein indicator until
the appearance of amethystine colour according to the Official Methods of the


V represented the volume of NaOH used and m was sample weight.


Enumeration of viable cell count (VCC)

Enumeration of L.casei was performed on the MRS-agar (Amyl media, Dandenong,
Australia) containing filter sterilized vancomycin in a final concentration of 0.35
M, was added to the liquid after autoclaving MRS-agar (DSM Co. catalogue).For
microbiological analyses, 5g of a yogurt sample was suspended in 45 mL sterile
peptone water (0.1%) and 10-fold (103 to 108) serially
diluted. By using pour plate technique, 1 ml of the appropriate dilutions was
cultured and the plates were incubated at 37?C for 72 h 21. Plates containing
30-300 colonies were counted and the results expressed as colony forming units
per gram (cfu? g) of the sample. 

Proteolysis activity by
o-Phthaladehyde (OPA)

µl of yogurt extract was added to 3 ml of OPA reagent (base on ?-
mercaptoethanol) 22 in a 5mL quartz cuvette and was mixed for 5 seconds.
After keeping it for 2 minutes at room temperature, the absorption level was
read by spectrometry (Beckman, America) at 340 nm. Distilled water was used
instead of yogurt sample as the blank. The amount of sample for proteolysis was
expressed based on measured peptides and free amino acids absorption 23. The peptide concentration was assessed against
tryptone standard.


of ACE inhibition Activity

of ACE-inhibition activity by yogurt was prepared following the protocol
described by Pihlanto 12.

According to this method, 125 ?l of FAPGG reagent was
added to 125 ?l of yogurt samples in the cuvette. The mixture was mixed
thoroughly and was incubated in water bath at 37?C for 15min.Rabbit long
extract (300 ?l in 50 mmol/l Tris-HCl) was added to the mixture. The absorption
of samples was measured at 340 nm (At0) then the plate was incubated at 37?C
for 10 min and the final absorption was measured (At10). Enalapril was used as
the standard. The determinations of ACE inhibition was carried out in
duplicate. The inhibitory activity was calculated as follows 23.


 Results and Discussion

Determination of pH and the acidity

According to Fig. 1 by increasing the amount
of Mentha.piperita extract, the pH of the sample was decreased from 4.2
to 3.5 and acidity was increased from 1 to 1.2 during 20 days of storage. It
seems that the presence of Mentha.piperita extract caused an increase in
the metabolic activity of yogurt bacteria 23 and it provides more substrates
for bacterial proteolysis and cell growth in presence of phenolic compounds
24. The number of free amino groups and peptides increased by hydrolyzing milk
proteins by bacteria proteinases and peptidases 3. However, by increasing the
amount of sodium caseinate, pH increased and the acidity decreased 25
probably it was due to as usage of carbohydrates by the microorganism and
producing more organic acids 26. On the other hand, calcium located in the
casein micelles is released by proteolysis and acidification of milk as a
result of lactic acid production during the fermentation process. So
antihypertensive characteristic of fermented milk may be explained by the high
concentration of free –Ca2+ in micelles.González and Nielsen 11, 27 reported when pH reached 4.3-4.6 showed an ACE < 50%. At the pH 6 the activity of cell-envelope proteinases and amino peptidases increased followed by decreased at lower pH values. Pan 28 reported that the –Ca2+ concentration was correlated with pH and ACE inhibitory. L.casei showed the highest ACE-inhibitory activity at low –Ca2+ concentrations. Our results was parallel to the results found in Gonzalez et al 11. Highest ACE inhibition was observed when the pH of yogurt reached 4.1 at 20th day of storage. However, other component in milk like concentration of bioactive peptides may affect the inhibition of ACE. It was observed that the amount of free amino acids and peptides in milk were lowered indicating that LAB started to degradation of milk protein and other proteins such as sodium caseinate which the process was found to be time-dependent 21. By terminating carbohydrates and also by increasing the number of liberated amino groups during fermentation 21, microorganisms started utilizing both of environment proteins and the organic acid in the period of time and caused increasing of pH and reducing acidity 29,30with time. Yogurt containing 4% sodium caseinate had a strong buffering capacity this explain why yogurt supplemented with sodium caseinate showed weaker capacity for post acidification during storage than others. Probiotic Cell Count The change in viable cell count of L.casei is presented in Figure 2. With prolonged storage period, the number of L.casei was reduced, then increased. This study showed that at the 4th day of storage, pH decreased followed by an increase in the 10th day of storage. By increasing the amount of sodium caseinate from 0 to 4% the number of L.casei increased, which could be due to increasing of the available substrate for the growth of bacteria 12. Lactic acid bacteria needs free amino acids more than milk natural protein for growth 21. Therefore LAB tends to milk proteins. Donkor et al 21 reported that the proteolysis was different between the LAB and it is dependent on time. Thus in the early stage of fermentation, the growth of bacteria was slow and increased by passing time. At the lack of free amino acids, yogurt culture should have a proteolytic capacity and provided free amino acids so the most important reason for LAB survival is protein hydrolysis system 9. Chen et al 3 reported the positive correlation between ACE-inhibition and free amino acids and the increase in titratable acidity and free amino acids was related to bacterial activities. In this work highest viable count was observed in pH 4.42 that was parallel the results found by Wang et al 9. In the initial hydrolysis of casein during fermentation, oligopeptides were produced by extracellular proteinases further hydrolysis of oligopeptides is necessary to fulfil cultural needs. Amirdivani et al 23 reported that the survival of the starter bacteria by increasing the amount of peppermint extract was increased. Because of limitation in the amount of environment lactose, the used starter bacteria should be applied in advance, which has resulted in  a reduction of the number of L.casei, but bypassing the time and using the other sources, and by synergistic influence of starter culture on probiotics growth 31, the number of L.casei was increased. Gonzalez et al 11 reported that L. casei produced the most potent peptides with an IC50 value of 0.47 mg/mL after 48 h fermentation. After fermentation, the yogurt contained sodium caseinate and peppermint extract at the cell density of 9.1 log cfu/mg, had pH of 4.42. Proteolysis evaluation Proteolysis is the most important biochemical process that takes place during fermentation and storage 1. During fermentation, milk proteins are hydrolyzed by LAB exopeptidase and lead to increasing free -NH2 groups whose amount can be determined by OPA method. The result of statistical analysis suggests that the effect of sodium caseinate and storage time and mutual effect of sodium caseinate and peppermint extract on yogurt proteolysis were significant (p<0.05). According to the figure 3a, bypassing storage time and increasing sodium caseinate, proteolysis increased. Based on figure 3b, at the low amount of peppermint extract, by increasing sodium caseinate, proteolysis increased but at the high amount of this extract, by adding to sodium caseinate, first, proteolysis increased and then decreased. The possible reason is that the extract brings about growing cellular metabolism and consequently increasing acidity 32. Therefore, the produced lactic acid has influenced yogurt proteins and led to protein denaturation and facilitating hydrolysis. However, by developing hydrolysis, the produced peptides and free amino acids cause to decrease the acidity. In the same way, by using up sugar materials, bacteria begin to consume the produced organic acids and peptides and as a result, the proteolysis decreased 33. With increasing sodium caseinate, the growth of L.casei increased due to the requirement for the growth available materials and intra and outer cellular enzymes 32, 33. These enzymes can hydrolyze active biologic peptides and bradykinin which are among the widely used enzymes at the food industry. Proteolysis provides necessary growth factors in the form of peptides and amino acids to enhance L. casei growth and survival and accordingly, proteolysis increases due to available materials for growth. ACE-inhibitory activity Figure 4 showed that at the early stage of storage period, by increasing the percentage of sodium caseinate, the ACE-inhibitory effect was significantly (p<0.05) increased and at the end of the period it was reduced. This may relate to the fact that as time passes, bioactive peptides from protein hydrolysis will be used by microorganisms. At 4th day of storage, the inhibition rate was high but by the 20th day of storage, it had reached a minimum amount. Our results are in agreement with those of results by Amirdivani et al 23. These results showed that peptides are produced with the less inhibitory property during fermentation, which they are broken down to more and smaller bioactive peptides in the middle of the maintenance period. During the storage time, the wide proteolysis of proteins causes the producing protein with less and smaller bioactive property up to 20 days 23. At 4th day of storage time, by increasing the amount of peppermint extract, the inhibition was not changed and followed by an increase in the 20th day of storage. As the soluble extract did not create any effect on ACE activity, this may have indirectly changed the proteins hydrolysis by effecting yogurt bacteria metabolism 23. ACE-inhibitory activity was increased because of the increasing of hydrolysis of proteins. The following prediction equation was obtained to ACE inhibition activity.                                                                  Which X1 is Sodium caseinate percentage, X2 is peppermint extract percentage and X3 is storage time (day).        These results showed that by increasing the amount of sodium caseinate and peppermint extract lead to increase the bacterial growth. Due to more proteolysis, the number of bioactive peptides and also the rate of ACE inhibition were elevated significantly (p< 0.05). During the storage time however bioactive peptides were hydrolyzed for bacteria usage and subsequently the rate of ACE inhibition was reduced. In this study, IC50 values were from 0.35 mg?ml to 0.12 mg?ml that agree with the results found in Leclerc et al 34. Samples containing 4% sodium caseinate and 0.4% peppermint extract and sodium caseinate together at 20th day of storage time had no detected ACE-inhibitory. The highest IC50 value was observed in yogurt supplemented with 4% peppermint extract (0.12 mg?ml).    Conclusion The present study addressed the changes of yogurts samples in the presence of sodium caseinate and peppermint extract. With the increase of sodium caseinate, the growth of L. casei increased that lead to increasing the production of bioactive substances and also ACE inhibitory activity but the inhibition didn't significantly influence using sodium caseinate and peppermint extract together during storage time. Utilization of sodium caseinate and peppermint extract is recommended, because of its improving effect on non-fat yogurts properties, notably texture, pH, L. casei viability, proteolysis and ACE inhibition. The results indicated that during storage time proteolysis increased in all samples containing peppermint extract. But in the high level of sodium caseinate ACE inhibition decreased during storage time. When sodium caseinate and peppermint extract were used together, no increase was found and sometimes the inhibition decreased.