Inclusion of Mentha piperita, Anethum graveolence and Ocimum basilicum in Yogurt and their effect on the Inhibition of Enzyme

Relevant to Hypertension and type-2 Diabetes

SHABBOO AMIRDIVANI

This thesis submitted in partial fulfilment of the Master of Science degree

Department of Biochemistry Institute of Biological Science University of Malaya 50603 Kuala Lumpur Session 2007/2008

ACKNOWLEDEGEMENT
I thank God for blessing me with the bravery, strength and perseverance, that enable me finished this project. Here, I would like to take the opportunity to express my deepest gratitude to those who made the success of this project possible. Associate Professor Dr. Ahmad Salihin Hj Baba, who has been sacrificing his time in providing me with the extensive guidance required to this project. My thanks to him may not be enough for what he had helped me. Words alone cannot express my deep thanks to my husband (Dr.Hossein Moshiri), who never failed to give me advices and encourage and supporting me each time. I cannot thank him enough for the kindly his helped. My greatest gratitude to my parents, for their never ending support and motivation. Their effort and care have given me courage and strength, thank you! Special thanks to my best friend Afsaneh Najarian, who guided me always. Thanks for her valuable advice as well as encouragement during my research. I wish the best for her. Finally, I would like to say thanks to all friends who helped and supported me to finish enable a successful complete of this thesis. Shabboo December, 2008

ABSTRACT
Medicinal herbs are widely used both in culinary and as therapeutic herbs in traditional medicine. The present study investigated the effects of inclusion of herbs into milk on yogurt formation and the effects on -amylase, -glucosidase and angiotensin I converting enzyme (ACE) activities. Pasteurised cows milk and starter culture mixture (Streptococcus thermophilus and Lactobacillus acidophilus) were incubated at 41C in the presence or absence of herbalwater extracts followed by storage at 4C. The addition of herbs into yogurt reduced pH at a faster rate than that in plain yogurt. pH 4.5 was achieved fastest for Anethum graveolenceyogurt (240 minutes) followed by Ocimum basilicum- and Mentha piperita-yogurts (both 300 minutes) and plain-yogurt (420 minutes). The TTA for these herbal-yogurts were higher than plain yogurt at all time points during the incubation. Both O. basilicum and M. piperita-yogurts had the same rate of TTA production whereas A. graveolence-yogurt had higher rate of TTA production (p<0.05) between the 180th- 240th minute of fermentation. All herbal yogurts had higher (p<0.05) antioxidant activity than plain yogurt. The OPA peptides in herbal yogurts increased by 28-36% by the 7th day of storage, with highest increase in M. piperita- followed by A. graveolence- and O. basilicum-yoghurts (33.4, 31.5, 30.2 mg/g respectively). All herbal yogurts showed higher anti-ACE activity than plain yogurt at respective storage periods. M. piperita-yogurt had the highest inhibitory effect on ACE activity throughout the storage period (84.9, 87.3, 74.0 and 63.6 % on day 0, 7, 14, 21 and 28 respectively). O. basilicumyogurts showed higher (p<0.05) antiACE activity than plain yogurt only on day 0 of storage. A. graveolence-yogurt however showed

stronger anti-ACE activity than O. basilicum-yogurt compared to plain-yogurt on all storage days except on day 28. Most potent effect on -amylase inhibition on day 7 of storage was shown by A. graveolence-yogurt followed by M. piperita and O. basilicum-yogurts (26.6, 37.3, and 42.0% respectively). All herbal-yogurts also inhibited -glucosidase, with highest inhibition on day 7 caused by A. graveolence-yogurt, M. piperita-yogurt and O. basilicum-yogurt (38.7, 22.6 and 15.7% respectively). The herbs studied modified yogurt formation with beneficial effects on enzymes important in the management of hypertension and diabetes mellitus.

ABSTRAK
Tumbuhan herba perubatan telah digunakan secara meluas dalam bidang masakan dan juga dalam perubatan tradisional. Kajian ini dilakukan untuk mengkaji kesan terhadap kehadiran ekstrak herba ke atas susu dalam penghasilan yogurt dan kesan terhadap enzim -amilase, glukosidase dan enzim pengubah angiotension I (ACE). Susu lembu terpasturasi dan campuran kultur bakteria (Streptococcus thermophilus dan Lactobacillus acidophilus) diinkubasi pada suhu 41C dengan kehadiran ekstrak herba(yogurt herba) atau tanpa kehadiran penambahan herba ke delam yogurt menurunkan pH ekstrak-air herba diikuti dengan penstoran pada suhu 4C. Pehambahan herba pH yogurt menurun dengan kadar yang lebih cepat berbanding dengan kontrol. Yogurt- Anethum graveolence telah menunjukkan indeks pH 4.5 paling cepat diikuti yogurt-Ocimum basilicum, dan yogurt Mentha piperita dan yogurt kawalan (240, 300, 300 dan 420 minit masing-masing). Kesemua yogurt herba menunjukkan kandungan TTA yang tinggi berbanding yogurt kawalan pada setiap bacaan semasa inkubasi. Yogurt O. basilicum dan yogurt M. piperita menunjukkan kadar penghasilan TTA yang sama manakala yogurt A. graveolence menunjukkan kadar penghasilan TTA yang tertinggi (p<0.05) pada jangkamasa diantara minit ke-180 hingga minit ke-240 waktu fermentasi. Kesemua yogurt herba menunjukkan aktiviti antioksidan yang tinggi (p<0.05) berbanding control. Peptida OPA dalam yogurt herba meningkat ke tahap 28-36% pada hari ke-7 tempoh simpanan. Tahap tertinggi ditunjukkan oleh yogurt- M. piperita seterusnya yogurt- A. graveolence- dan yogurt O. basilicum (33.41, 31.47, 30.22 mg/g masing-masing). Kesemua yogurt herba juga menunjukkan aktiviti anti-ACE yang tinggi berbanding yogurt kawalan sepanjng tempoh simpanan. Yogurt M. piperita mempunyai

tahap kesan perencatan yang tinggi pada aktiviti ACE sepanjang tempoh masa simpanan (84.9, 87.3, 74.0 dan 63.6 % pada hari ke 0, 7, 14, 21 dan 28 masing-masing). Yogurt O. basilicum menunjukkan aktiviti anti-ACE tinggi yang berbanding yogurt kawalan hanya pada hari ke 0 tempoh simpanan. Bagi yogurt A. graveolence pula, kesan aktiviti anti-ACE yang ditunjukkan amat tinggi daripada yogurt O. basilicum berbanding yogurt kawalan pada setiap masa simpanan kecuali pada hari ke 28. Kesan perencatan -amylase yang paling berkesan adalah pada hari ke 7 tempoh simpanan yang ditunjukkan oleh yogurt A.graveolence, diikuti yogurt M.piperita dan yogurt O.basilicum (26.6, 37.2, and 42.0% masing-masing). Kesemua yogurt herba juga menunjukkan kesan perencatan terhadap -glucosidase dengan perencatan tertinggi pada hari ke7 ditunjukkan oleh yogurt A. graveolence, yogurt M. piperita and yogurt O. basilicum (38.7, 22.6 dan 15.7% masing-masing). Herba yang dikaji mempengaruhi pembentukan yogurt dengan kesan bermanfaat ke atas enzim-enzim penting dalam pengurusan hipertensi dan diabetes mellitus.

Table of Content Acknowledgement. Abstarct Abstrak. Table of Content Abbreviation. List of figure.. List of tables I II IV VI XI XII XIII

CHAPTER 1 Introduction.......... 1

CHPTER 2 2.1Yogurt................................ 2.1.1 Nutritional value of yogurt............................ 2.1.1.1 Vitamin B...... 2.1.1.2Lactose................................... 2.1.1.3 Protein........... 2.1.1.4 Mineral.......................... 2.1.1.5 Lipids 2.1.2 Health benefit of yogurt... 2.1.2.1 Anti carcinogenic activity 2.1.2.2 Reduced risk of colon cancer...... 2.1.2.3 Reduced the risk of osteoporosis..... 2.1.2.4 Reduced incidence hypertension..... 2.1.2.5 Discouraging vaginal infection....................................................................... 4 4 4 5 5 6 7 7 7 8 8 8 9

2.1.3 Type of yogurt.............................................................................................. 2.2 Probiotic.................................... 2.2.1 Type of probiotic.......... 2.2.1.1 Lactobacillus acidophilus .............. 2.2.1.2 Bifidobacteriun.... 2.2.1.3 S. thermophilus. 2.2.2 Health benefit of probiotic.... 2.2.2.1 Nutrient synthesis and bioavailability.......... 2.2.2.2 Immune system.... 2.2.2.3 Lactose intolerance...... 2.2.2.4 Allergy........ 2.2.2.5 Cancer..................... 2.3 Diabetes.... 2.3.1 Complication associated with diabetes mellitus....... 2.3.2 Prevention and treatment diabetes....... 2.3.3 Economic costs of diabetes.. 2.3.4 Side effects of drug and precautions.... 2.3.5 Traditional plant medicines as treatments for diabetes 2.3.5.1 Herbal extracts as anti-diabetic. 2.4 Hypertension.. 2.5 Ocimum basilicum...... 2.5.1 Phytochemical components of O. basilicum 2.5.2 Health benefits of O.basilicum... 2.6 Mentha piperita.. 2.6.1 Phytochemical content of M. piperita

10 11 11 11 12 12 13 13 14 14 14 15 15 17 18 19 20 20 21 22 25 25 26 27 28

2.6.2 Health Benefits of M. piperita... 2.7 Anethum graveolence... 2.7.1 Phytochemicals in A. graveolence. 2.7. 2 Medicinal Uses........

28 29 29 30

CHAPTER 3 3.1 Materials... 3.1.1 Herbs. 3.1.2 Starter Culture........ 3.2 Methods.. 3.2.1 Water extraction of herbs........ 3.2.2 Yogurt. 3.2.2.1 Preparation of starter culture. 3.2.2.2 Preparation of yogurt 3.2.3 pH and TTA. 3.2.4 Preparation of yogurts extracts.. 3.2.5 Antioxidant activity determination by 1,1-Diphenyl-2-Picrylhydrazyl (DPPH) radical inhibition assay (Apostolidis, et al. 2006).. 3.2.6 Total Phenolic assay..... 3.2.7 O-Pthaldialdehyde Assay (OPA) (Goodno et al 1981)... 3.2.7.1 Preparation of OPA reagent.... 3.2.7.2 OPA assay (Goodno et al 1981).. 3.2.7.3 Tryptone standard curve (Goodno et al 1981) 3.2.8 ACE inhibition assay (Vermeirssen et al. 2002)..... 3.2.8.1 Preparation of rabbit lung extract (Vermeirssen et al. 2002) 33 34 35 35 36 36 37 37 31 31 31 31 31 32 32 32 32 33

3.2.8.2 Preparation of standard rabbit lung (Vermeirssen et al. 2002) 3.2.8.3 Preparation of ACE reagent (Vermeirssen et al. 2002) 3.2.8.4 Preparation of Enalapril (Vermeirssen et al. 2002).. 3.2.8.5 Measurement of rabbit lung extracts ACE activity (control).. 3.2.8.6 Measurement of standard rabbit lung (sigma) ACE activity 3.2.8.7 Measurement of rabbit lung extract ACE activity in the presence if inhibitor (Enalapril)..... 3.2.8.8 Measurement of anti-ACE activities of water yogurt extract 3.2.8.9 Calculation of ACE inhibition activity (Vermeirssen et al. 2002).... 3.2.9 -Amylase inhibition assay (E. Apostolidis et al. 2007)........ 3.2.9.1 -Amylase enzyme solution... 3.2.9.2 Sodium phosphate buffer (0.02 M), pH 6.9 with .006 M sodium chloride..................................................................................................................... 3.2.9.3 Dinitrosalicyclic acid (DNSA) reagent.. 3.2.9.4 1% starch solution ........ 3.2.9.5 -Amylase inhibition assay.... 3.2.10 -Glucosidase inhibition ( E. Apostolidis et al. 2007)............ 3.2.10.1 -Glucosidase enzyme solution.... 3.2.10.2 0.1M potassium phosphate buffer (pH 6.90)...... 3.2.10.3 5mM p-nitrophenyl--D-glucopyranoside substrate solution... 3.2.10.4 -Glucosidase Inhibition Assay ...... 3.2.11 Calculation of IC50 for enzyme inhibition activity...... 3.2.12 Statistical analysis.......

38 38 38 39 39

40 40 40 41 41

41 42 42 42 43 43 43 44 44 45 45

CHAPTER 4 4.1 Change of yogurts pH...... 47

4.2 Total titratable acid (TTA) of yogurts.. 4.3 Total phenolic content (TPC) in yogurts..... 4.4 Antioxidant capacity of yogurts.. 4.5 Concentration of OPA peptides 4.6 Angiotensin-1 converting enzyme (ACE) 4.7 Alpha-amylase inhibition.. 4.8 Alpha- glucosidase inhibition......

48 49 50 52 53 55 57

4.9 Regression values of correlation between TPC and anti-diabetic enzyme inhibition activities, and OPA peptides and ACE activity...............................................................

CHAPTER 5 5.1 Effect of herbs (M. piperita, O. basilicum and A. graveolence) on yogurt fermentation...................................................................................................................... 5.2 Total phenolic content in herbal-yogurts........ 5.3 Antioxidant activities of herbal-yogurts......... 5.4 Proteolysis and changes in OPA peptides in herbal-yogurts...... 5.5. Inhibition of angiotensin-1 converting enzyme (ACE) activity by herbal-yogurts 5.6 Inhibition of -amylase activity by herbal-yogurts. 5.7 Inhibition of -glucosidase activity by herbal-yogurts. 5.8 General discussion. 5.9 Conclusion... References.....

61 63 64 66 67 68 69 70 75 76

ABBREVIATION
% & C etc g h HCL i.e. LAB mg Min ml nm vs l g ACE-I A.graveolence M.piperita O.basilicum OPA NaOH BF Percentage and degrees Celsius et cetera gram(s) hour(s) Hydrochloric acid For example Lactic Acid Bacteria milligram(s) minute(s) millilitre nanometer versus microliter microgram(s) Angiotensin-I Converting Enzyme Anethum graveolence Mentha piperita Ocimum basilicum O-phthalaldehyde Sodium hydroxide before fermentation

LIST OF FIGURES
Figure 3.1: Typical standard curve of gallic acid for the estimation of total phenolic in yogurts...............................................................................................................35 Figure3.2: Typical Standard curve of tryptone for OPA peptides estimation.........36 Figure 4.1: Changes of pH of yogurts during lactic acid bacteria fermentation of milk in the presence of herbs at 41C......................................................................................46 Figure 4.2: Changes of total titratable acid (TTA) in yogurt during lactic acid bacteria fermentation of milk in the absence or presence of herbs at 41C............................47 Figure 4.3: total phenolic content of water extract of milk + herbs before fermentation, fresh yogurts (0 day) and yogurts stored (4C) up to 28 days...................................48 Figure 4.4: Inhibition (%) of DPPH scavenging activity by water extract of milk + herbs before fermentation, fresh yogurts (0 day) and yogurts stored (4C) up to 28 days..49 Figure 4.5: OPA peptides in water extract of milk + herbs before fermentation, fresh yogurts (0 day) and yogurts stored (4C) up to 28 days.............................................51 Figure 4.6: Effect of refrigerated storage (4C) of yogurt water extracts for 28 days on angiotensin-1 converting enzyme (ACE) inhibition (%)............................................52 Figure 4.7: Angiotensin converting enzyme IC50 of water extract of milk + herbs before fermentation, fresh yogurts (0 day) and yogurts stored (4C) up to 28 days............53 Figure 4.8: Effect of refrigerated storage (4C) of yogurt water extracts for 28 days on -amylase inhibition (%).......................................................................................... 54 Figure 4.9: -amylase IC50 of water extract of milk + herbs before fermentation, fresh yogurts (0 day) and yogurts stored (4C) up to 28 days..........................................55 Figure 4.10: Effect of refrigerated storage (4C) of yogurt water extracts for 28 days on glucosidase inhibition (%)..................................................................................... 55 Figure 4.11: -glucosidase IC50 of water extract of milk + herbs before fermentation, fresh yogurts (0 day) and yogurts stored (4C) up to 28 days....................................57

List of Tables

Table2.1 Type of yogurt........................................................10 Table4.1 Regression values of correlation between TPC and anti-diabetic enzyme inhibition activities, and OPA peptides and ACE activity..........................58

Introduction
Yogurt is a widely consumed functional food due to its good taste and nutritional properties. It is rich in potassium, calcium, protein and vitamin B and has beneficial effects on human health by supplying pre-biotics and pro-biotics bacteria. It helps to strengthen the immune system, improves lactose digestion and gastrointestinal conditions including lactose intolerance, constipation, diarrhoea, colon cancer, inflammatory bowel disease and allergies (FitzGerald et al., 2004). Apart from the nutritional benefits of consuming conserved milk, yogurt eating has been associated with a number of health benefits including but not limited to improvement in lactose metabolism, anti-mutagenic properties, anti-carcinogenic properties, reduction in serum cholesterol and management of hypertension, anti-diarrhoeal properties, immune system stimulation, improvement in inflammatory bowel disease and suppression of Helicobacter pylori infection (Kurmann & Rasic, 1991; Shah, 2000). The beneficial health effects of yogurt have been partly linked to the proteolysis products produced during fermentation and storage. In particular, a group of peptides can lower the blood pressure in hypertensive patients (FitzGerald et al., 2004). One of the most important intermediary factors for controlling hypertension is the action of the angiotensin converting enzyme-I (ACE-I) (Hernadez-Ledesma et al. 2003). Angiotensin I-converting enzyme (ACE-I) is an important enzyme involved in maintaining vascular tension. ACE activates a histidyl-leucine dipeptide called angiotensin I into a potent vasoconstrictor called angiotensin II (Skeggs et al. 1956). Angiotensin II also stimulates the synthesis and release of aldosterone, which increase blood pressure by promoting sodium retention in the distal tubules (Lieberman, 1975). Inhibition of ACE is considered a useful

therapeutic approach in the treatment of high blood pressure in both diabetic and nondiabetic patients (Johnston & Franz 1992; Erdos & Skidgel 1987). Recent studies indicate that flavonoid-rich food and plants have the ability to inhibit ACE-I activity, both in vitro and in vivo (Actis-Goreta et al. 2003; Kwon et al.2006). The consumption of selected flavonoid-rich foods may mimic synthetic ACE-I inhibitors and provide health benefits, but without adverse side effects. These plants also contain phenolic phytochemicals. These are secondary metabolites of plant origin that constitute one of the most abundant groups of natural metabolites and form an important part of both human and animal diets (Bravo 1998; Crozier et al. 2000; Vattem et al. 2005). Recent studies have shown that phenolic phytochemicals have high antioxidant activity and certain therapeutic properties (Shetty et al. 2005), including anti-diabetic and anti-hypertension activity (Kwon et al. 2006). The present studies investigate commonly used herbs (Ocimum basilicum (basil), Mentha piperita (peppermint) and Anethum graveolence (dill)) in Iran on yogurt formation. These plants contain bioactive compounds which affect myriads of activity including anti-bacterial, antioxidant, anti-diabetic and anti-mutagenic. O. basilicum of the Family Lamiaceae is used in traditional medicine and as a culinary herb is a well-known source of flavouring principles (Javanmardi, 2003). A number of phenolic compounds with strong antioxidant activity have been identified in these plant extracts (Nakatani, 1997) and have been implicated having anti-aging, anti-cancer, anti-viral, and anti-microbial properties (Manosroi, 2004). M. piperita, a hybrid mint of watermint (Mentha aquatica) and spearmint (Mentha spicata) contains biologically active constituents and has high menthol, menthone and menthyl esters and is widely used against upset stomachs, inhibits the growth of certain bacteria, and can help soothe and relax muscles when inhaled or applied to the skin (Sharma & Tyagi 1991, Shasany et al. 2000). A. graveolence which

contains myristicin, apiol and dillapiol is a medicinal plant regularly used as an antispasmodic, carminative, diuretic and stimulant for lactation (De Rougemont et al., 1989). A. graveolence also acts as an anti-bacterial agent and offers protection from carcinogens and free radicals (Kwon et al. 2006).A. graveolence is known medicinally for its soothing effects on distress stomach and is safe for children and adults (Kwon et al. 2006). The consumption of yogurt in recent decade has increased rapidly largely due to the fact that this dairy product meets many consumer dietary needs. It is a ready-to-eat food which can be designed to fulfil variety of specifications: rich in fat right down to low in fat but rich in nutritional components. Attempts to make the fermented milk more palatable and nutritious have recently extended in the inclusion of rare fruits, collagen and prebiotics in the making of yogurts. In this regard the inclusion of healthy herbs is expected to enhance the nutritional values of yoghurt and as such enhanced existing perceived values of herbal-yoghurts. To date very little is known about the changes that can occur to yogurt when herbs are also present during milk fermentation. This thesis reports the changes occurring in yogurt as a result of the presence of M. piperita, A. graveolence or O. basilicum during probiotic fermentation of milk.

2.1 Yogurt
Yogurt is coagulated milk traditionally obtained by lactic acid fermentation through the action of lactic acid bacteria Lactobacillus caseie and Streptococcus thermophilus (Orihara et al., 1998). The nutritional value of yogurt is made up of the nutrients of the milk and the nutrient among metabolites produced during the fermentation by lactic acid bacteria (Yukuchi et al,

1992). An important development of yogurt making is a new generation of professed bio-yogurt which has very different microflora from the traditional product. This type of yogurt may contain L. acidophilus, L. casei, and Bifidobacterium bifidum, which is in fact natural microflora of the human intestine. These bacteria are known as probiotic because they have a beneficial effect on intestinal function and promote good health (Sanders,1999) by colonizing the distal portion of the small intestine (Lactobacilli) and form one of the dominant groups in the colon (bifidobacteria) (HayaKawa,1992).

2.1.1 Nutritional value of yogurt

Yogurt hasss generally been considered as an excellent source of high quality protein, calcium, potassium, phosphorus, magnesium, zinc and riboflavin, niacin, vitamin B6 and vitamin B12 (Shah, 2007).

2.1.1.1 Vitamin B

LAB species need vitamin B for growth, but some cultures are capable to synthesize vitamin B (Buttress, 1997). An example of a vitamin B that is utilized by LAB is vitamin B12 (Reddy et al., 1976). Folate is a B vitamin that some LAB species synthesize (Crittenden et al., 2003). The major form of folate present in milk is 5-methyl-tetrahydrofolate (Wigertz et al, 1996). Studies in bacterial isolates from various species used for milk fermentation and yogurt production examined for their ability to synthesize or utilize folate (Crittenden et al., 2003) showed that S. thermophilus and Bifidobactereia was folate producer, while Lactobacilli reduce

folate from the milk media. A combination of folate-producing cultures also showed an even greater folate of the final fermented product (Shah, 2007).

2.1.1.2 Lactose

Yogurt is an exclusive source of the lactose in human diets. Lactose (O--Dgalactopyranosyl-(14)-D-glucopyranose; disaccharide) is a milk sugar that occurs naturally only in milk (Voet and Voet2004). Lactose however has to be hydrolyzed by the -galactosidase (lactase) into glucose and galactose before absorption and utilization as energy sources. The hydrolysis of lactose during fermentation resulted in significant reduction of milk lactose to 7080% (Bourlioux and Pochart, 1988). This make yogurt to be more tolerated than milk by people with lactose mal-digestion (lactose intolerance) (Vesa et al., 2000). L. caseie and S. thermophilus in yogurt also expressed functional lactase, the enzyme that break down lactose (Goodenough & Kleyn, 1976).
2.1.1.3 Protein

Generally the protein content of yogurt is higher than milk due to the addition of non-fat dry milk during processing and concentration, which increases the protein content of the product. Nevertheless, the protein from yogurt is more easily digested, as yogurt bacteria pre-digest the milk proteins in yogurt (Shahani & Chandan, 1979). Thus there is higher content of free amino acids, especially proline and glycine, in yogurt more than in milk. The activity of proteolytic enzymes and peptides is preserved throughout the shelf life of the yogurt. L. caseie have a much higher proteolytic activity during milk fermentation and storage than does S. thermophilus, as indicated by elevated concentrations of peptides and free amino acids after milk fermentation

(Beshkova et al.,1998). During fermentation, both heat treatment and acid production result in finer coagulation of casein, which may also contribute to the greater digestibility of yogurt than of milk. Both the caseins and the whey proteins in yogurt are rich source of the essential amino acids (Bissonnette & Jeejeebhoy , 1994).

2.1.1.4 Minerals

In addition to being a good source of protein, yogurt is also a rich source of calcium and phosphorus. In fact, dairy products such as milk, yogurt, and cheese provide highly available calcium in diet. Because of the lower pH (4.5-4.8) of yogurt compared with that of milk, calcium and magnesium are present in yogurt mostly in their ionic forms (Shah, 2007).

2.1.1.5 Lipids

Milk fat also goes through biochemical changes during fermentation. Minor amount of free fatty acids are released as a result of lipase activity. For instance yogurt was shown to have a higher concentration of conjugated linoleic acid (Shantha et al.,1995). The fat of yogurt is more bio-available compared to milk although the increas in free saturated fatty acid is small (Tannous & Merat, 1969).

2.1.2 Health benefit of yogurt

There are many health benefits of yogurt. Lactic acid aids in calcium absorption and digesting some of the lactose for people with lactose intolerance (Salwa et. al., 2004; Laye et. al., 1993; Tarakci & Kucukoner, 2004; Burton & Tannock, 1997). Yogurt also acts as an antibiotic (Laye et. al., 1993), protects against gastrointestinal upset (Salwa et. al., 2004; Laye et. al., 1993), decreased risk of cancer (Salwa et al., 2004), lower blood cholesterol especially low density lipoprotein cholesterol (Salwa et al., 2004; Akalin et. al., 1997; Rodas et. al., 1996), help the body assimilate protein, calcium and iron (Salwa et. al., 2004). The organism in yogurt also can produce some B vitamins which are needed by body (Davis, 1970). Collectively, these contribute to a high level of nutrition and contribute to the strengthening of the immune system (Salwa et. al., 2004; Laffineur et. al., 1996; Kot et. al., 1996).

2.1.2.1 Anti carcinogenic activity

The anti tumour action of probiotics is attributed to the inhibition of carcinogens and procarcinogens, specifically by the inhibition of bacteria (reduction of the intestinal pH to reduce microbial activity) that convert procarcinogens (Gilliland et al., 1996) and activation of the hosts immune system (Rasic and Khurmann, 1978). Kailasapathy and Rybka (1997) reported that the intake of yogurt and fermented milks containing probiotic bacteria inhibited tumour formation and proliferation.

2.1.2.2 Reduced risk of colon cancer

Yogurt intake is associated with significant decreased risk of colon cancer (Peter, 1992). Calcium may reduce the risk of colon cancer, particularly as a result of increased dietary intake of low fat, calcium-rich dairy products such as yogurt (Rodas, et al., 1996).

2.1.2.3 Reduced the risk of osteoporosis

Calcium-rich yogurt can help slow bone loss, thereby preserving bone mass and lessen the effects of osteoporosis among the elderly, especially in post-menopausal women (Orihara et.al. 1998).

2.1.2.4 Reduced incidence hypertension

Calcium, potassium, and magnesium are present in milk in significant amounts. These minerals have been shown to reduce hypertension or high blood pressure, which increases the risk of heart disease, stroke, and kidney disease. Calcium-rich diet also helps regulate blood pressure in women during and after pregnancy (Rodas, et al., 1996).

2.1.2.5 Discouraging vaginal infection

Yeast vaginal infection is a common problem for women with diabetes. L. acidophilus, a strain of friendly bacteria, is an integral part of normal vaginal flora. Lactobacilli prevent overgrowth of unfriendly bacteria and Candida. They produce lactic acid, which acts like a natural antibiotic. On the other hand, these friendly bacteria also compete with other organisms

for the utilization of glucose. The production of lactic acid and hydrogen peroxide by lactobacilli helps to maintain the acidic pH needed for healthy vaginal flora (Laye, et al., 1993). In summary the highest quality yogurt available in the market contains high live bacteria that provide a host of health benefits. These live bacterial cultures may help to fortify the immune system, availability and reduce formation of carcinogenic and hence increase life expectancy. Increased yogurt consumption, particularly in immune compromised populations such as the elderly, may enhance the immune response, which would in turn increase resistance to immune-related diseases (Shah. 2000).

2.1.3 Types of yogurt

Yogurt products come in a wide variety of flavours, forms and texture according to the method of fermentation and yogurt presentation. These are briefly summarised in the following table (Orihara et.al. 1998). Table2.1)Type of yogurt Types Set Yogurt Yogurt preparation This is the most common type using no additive or varieties of stabilizers such as sugar, gelatine or pectin. This yogurt is incubated and cooled in the final package and is characterised by a firm jelly texture. Stirred Yogurt Concentrated Yogurt Does not have a jelly-like gel texture, but is highly viscous semi fluid. Yogurt is concentrated by boiling off some of the water; this is often done under the vacuum to reduce the temperature required. Protein is denatured and produced rough and gritty texture

Drink Yogurt Frozen Yogurt

It is even softer than stirred yogurt. It is a general term for fermented milks in liquid form Yogurt is most commonly stirred with emulsifiers and stabilizers and frozen in freezer like ice cream. Three types can be distinguished by overrun and storage temperature, hard (-25 C), soft (0 to -6C), and mousse (<0C).

Dried/Instant Yogurt

Frozen dry or sprayed dry to make processed powder. There are two types of dried yogurt, one type contain live bacteria, and is incubated with milk for several hours;the other type is instant yogurt, which forms a gel in a short time due to the use of stabilizers. The flavours added in yogurt usually in the form of puree or as whole fruit in syrup

Flavoured Yogurt

2.2 Probiotics
Probiotics are live microbial food supplements or components of bacteria, which have been shown to have beneficial effects on human health. They are perceived to exert such effects by changing the composition of the gut micro flora. Probiotic bacteria such as L. acidophilus, Bifidobacterium spp. and L. casei have long safe history in the manufacture of dairy products and are also found as a part of gastrointestinal micro flora (Shah, 2007). Several factors have been claimed to affect the viability of probiotic cultures in fermented milk products. Acidity, pH and hydrogen peroxide have been identified to have an effect during manufacture and storage of yoghurt Other factors, such as temperature of storage, oxygen content, concentrations of lactic and acetic acids have been presumed to affect the viability of probiotic organisms in yogurt (Lankaputhra & Shah, 1998).

2.2.1 Type of probiotics

2.2.1.1 Lactobacillus acidophilus

Lactobacillus gets its name from lacto, meaning milk and bacillus meaning rod-like in shape and acidophilus meaning acid-loving. This bacterium thrives in more acidic (pH 3-4) environments than most related micro organisms (pH 4-5 or lower) and grows best at 45C. L. acidophilus occurs naturally in the human and animal gastrointestinal tract, mouth, and vagina and protect against some unhealthy organisms. L. acidophilus breakdown nutrients and produces lactic acid, hydrogen peroxide and other by products that make the environment hostile for undesired organisms (Myers, 2007).

2.2.1.2 Bifidobacterium

Studies on Bifidobacterium was first dicovered by Tisser in 1900 when he discovered a rod later called Bacillus bifidus communis during microscopic observations of the intestinal microflora in infants (Kander, 2004). Bifidobacterium rods constitute autochtonic microflora of the alimentary tract, are among the first bacteria to colonize germ-free digestive tracts of newborns, and dominate in the large intestine microflora during lactation. Bifidobacteria are immobile, non-sporing and catalase-negative microorganisms. The optimum conditions for their development are a temperature of 37C and pH 5.6 - 6.4 . Most of them are anaerobic organisms, but particular species may differ in their sensitivity to oxygen (Kander, 2004). The growth of the majority of bifidobacteria is strongly inhibited by ampicillin, chloramphenicol, erythromycin, G penicillin and lincomycin (Piard et al. 1992). Bifidobacterium rods ferment glucose producing acetic and lactic acids (L+). Apart from these acids, they can also produce small quantities of

formic acid, ethanol, succinic acid, diacetyl, 2-pirolidon-5-carboxyl acid and bacteriocins (Kander 2004)
2.2.1.3 S. thermophilus

Streptococcus thermophilus is a gram-positive facultative anaerobe. It is a cytochrome-, oxidase- and catalase-negative organism that is nonmotile, non-spore forming and homofermentative. S. thermophilus is an alpha-hemolytic species of the viridans group and is also classified as lactic acid bacteria (LAB). S. thermophilus has an important role as a probiotic, alleviating symptoms of lactose intolerance and other gastrointestinal disorders (Shah, 2007).

2.2.2 Health benefit of probiotics

The consumption of probiotics are useful in the treatment of many types of diarrhoea, including antibiotic-associated diarrhoea in adults, travellers diarrhoea, and diarrhoeal diseases in young children caused by rotaviruses (Isolauri et al,. 1991). Since diarrhoea is a major cause of infant death worldwide and can be incapacitating in adults, the widespread use of probiotics could be an important, non-invasive means to prevent and treat these diseases, particularly in developing countries. Probiotic bacteria also preserve intestinal integrity and mediate the effects of inflammatory bowel diseases, irritable bowel syndrome, colitis, and alcoholic liver disease (Kruis, et al,. 1997). In addition, lactic acid bacteria may improve intestinal mobility and relieve constipation, particularly in seniors (Seki et al,. 1978).

2.2.2.1 Nutrient synthesis and bioavailability

Fermentation of food with lactic acid bacteria has been shown to increase folic acid content of yogurt, bifidus milk and kefir and to increase niacin and riboflavin levels in yogurt, and vitamin B12 in cottage cheese and vitamin B6 in Cheddar cheese (Alm, 1982). In addition to nutrient synthesis, probiotics may improve the digestibility of some dietary nutrients such as protein and fat (Shahani et al,. 1984).

2.2.2.2 Immune system

Probiotics can enhance both the specific and non-specific immune response, possibly by activating macrophages, increasing levels of cytokines, increasing natural killer cell activity, and/or increasing levels of immunoglobulins (Sanders, 1999).

2.2.2.3 Lactose intolerance

Lactic acid bacteria, such as S. thermophilus, L. casei and other lactobacilli in fermented milk products can improve symptoms of lactose intolerance by providing bacterial lactase to the intestine and stomach. Because lactose intolerance affects almost 70% of the population worldwide, consumption of these products may be a good way to incorporate dairy products and their accompanying nutrients into the diets of lactose intolerant individuals (Sanders, 1999).

2.2.2.4 Allergy

Probiotics may exert a beneficial effect on allergic reaction by improving mucosal barrier function. In addition, probiotic consumption by young children may beneficially affect immune

system development. Probiotics such as Lactobacillus may be helpful in alleviating some of the symptoms of food allergies such as those associated with milk protein (Majamaa, 1997). Probiotic consumption may thus be a means for primary prevention of allergy in susceptible individuals.

2.2.2.5 Cancer

Probiotic bacteria may reduce colon cancer risk by reducing the incidence and number of tumors. One clinical study showed an increased recurrence-free period in subjects with bladder cancer (Aso and Akazan, 1992).

2.3 Diabetes
Hyperglycemia is a condition characterized by an abnormal excess of sugar in the blood. Elevated postprandial blood glucose levels are widely recognized as one of the earliest disease markers in the prediction of subsequent microvascular and macrovascular complications that can progress to full symptomatic type 2 diabetes (T2DM) (Ratner 2001; Zimmerman 2001; Fonseca, 2003). The two major forms of diabetes are Type 1 (insulin-dependent diabetes mellitus) and Type 2 (noninsulin-dependent diabetes mellitus). Both Type 1 and Type 2 diabetes share one central feature: elevated blood sugar (glucose) levels. In Type 1, specifically, there is insufficient insulin secretion, a hormone produced by the pancreas (Dicarli et al. 2003). Type II diabetes is the most common form of diabetes, accounting for 90% of cases, and is usually characterized by an abnormal rise in blood sugar right after a meal, called postprandial hyperglycemia. Chronic

hyperglycemia is associated with significant long-term dysfunction and failure of various organs, especially the kidneys, eyes, nerves, heart and blood vessels (Canadian Diabetes Association 2003). The major source of blood glucose is dietary carbohydrates, such as starch, which are hydrolyzed by -glucosidases and pancreatic -amylase, in order to be absorbed by the small intestine. An effective strategy for Type 2 diabetes management is the inhibition of glucosidases and pancreatic -amylase (Krentz & Bailey2005). Intestinal -glucosidase inhibition, which delays the absorption of glucose following starch and sucrose conversion, moderates the postprandial blood glucose elevation, and thus mimics the effects of dieting on hyperglycemia (Bischoff 1994). -Glucosidase inhibitors currently used for diabetic patients are acarbose and miglitol, which result in the reduction of intestinal absorption of sugars in humans (Rybka et al. 1998; Inzucchi 2002). Chronic -amylase inhibition may be useful to treat Type 2 diabetes and obesity (Koike et al. 1995). -Amylase acts upon large polysaccharides (starch) at internal bonds (McCarter & Withers, 1994). Natural -amylase inhibitors offer an attractive therapeutic approach to the treatment of postprandial hyperglycemia by ultimately slowing glucose release from starch. However, excessive -amylase inhibition, resulting from certain -glucosidase inhibitors (acarbose), has certain side effects, such as abdominal distention, flatulence and possibly diarrhoea (Horii et al. 1987). Natural -amylase and -glucosidase inhibitors from plants have lower inhibitory effect against -amylase activity and a stronger inhibitory activity against -glucosidase, which therefore can be used as effective therapy for postprandial hyperglycemia with reduced side effects (Kwon et al. 2006). There are more than 180 million people worldwide who have diabetes and 90% of these suffer from Non-insulin-dependent Type II diabetes (World Health

Organization Website, 2006). The World Health Organization (WHO) estimates that the number of people with diabetes will double by 2030 (World Health Organization Website 2006). The WHO also states that Type II diabetes can be prevented by physical activity, healthy eating and prevention of obesity (World Health Organization Website 2006). Phenolic compounds are known to interact with proteins and can inhibit enzymatic activity (Dawra et al. 1988). In fact, many medicinal plant and herbal extracts have been found to inhibit the enzymatic activity of -glucosidase and -amylase (Kim et al, 2000; Grover et al, 2002; McCue and Shetty, 2004; McCue et al, 2004). Dietary -glucosidase and -amylase inhibitors that act in the gut by inhibiting the enzymatic breakdown of starch, soluble carbohydrates and their derived and digested products have been identified as a potentially natural and safe approach for controlling hyperglycemia through modulation (i.e., decrease) of meal-derived glucose absorption (Fonseca, 2003).

2.3.1 Complications associated with diabetes mellitus

Cardiovascular disease is responsible for between 50% and 80% of deaths in people with diabetes. Risk factors for heart disease in people with diabetes include high blood pressure, high serum cholesterol, obesity and smoking. Recognition and management of these conditions may delay or prevent heart disease in people with diabetes. Diabetic neuropathy is probably the most common complication. Studies suggest that up to 50% of people with diabetes are affected to some degree. Major risk factors of this condition are the level and duration of elevated blood glucose. Neuropathy can lead to sensory loss and damage to the limbs. It is also a major cause of impotence in diabetic men (Tiwari & Rao, 2002).

Diabetic retinopathy is a leading cause of blindness and visual disability. Research findings suggest that, after 15 years of diabetes, approximately 2% of people become blind, while about 10% develop severe visual handicap. Diabetes is among the leading causes of kidney failure, but its frequency varies between populations and is also related to the severity and duration of the disease. Diabetic foot disease, due to changes in blood vessels and nerves, often leads to ulceration and subsequent limb amputation. Diabetes is the most common cause of non-traumatic amputation of the lower limb.

2.3.2 Prevention and treatment diabetes

Primary prevention, healthy diet and regular physical activity, protects susceptible individuals. It has an impact by reducing or delaying both the need for diabetes care and the need to treat diabetes complications. It should be emphasized particularly in the poorest regions of the world where resources are severely limited. Secondary prevention includes early detection and good treatment. The treatment of high blood pressure and raised blood lipids, as well as the control of blood glucose levels, can substantially reduce the risk of developing complications and slow their progression. Large, population-based studies in China, Canada, USA and several European countries suggest that even moderate reduction in weight and half an hour of walking each day reduced the incidence of diabetes by more than one half in overweight subjects with mild impaired glucose tolerance.

2.3.3 Economic costs of diabetes

WHO figures show that about 80% of people with Type II diabetes are from low and middle income countries (World Health Organization Website 2006). Therefore there is a need for low cost and easily available methods for management of diabetes in order to improve the standard of living of most diabetes patients. Because of its chronic nature, the severity of its complications and the means required to control them, diabetes is a costly disease, not only for affected individuals and their families, but also for the health systems. In WHOs Western Pacific region a recent analysis of health care expenditure has shown that 16% of hospital expenditure was for people with diabetes. In the Republic of the Marshall Islands, this figure was 25% whereas 20% of "offshore expenditure" on health by Fiji was for diabetes-related complications - instances where facilities for care were not available in Fiji, so patients had to travel elsewhere. These represent considerable sums for countries who can still afford such massive expenditure on preventable conditions (WHO 2000). Although drug treatment is the first line of action for acute cases, non-drug therapies, i.e. lifestyle changes which include a regimen of physical activity, dietary adaptation and patient education must be the action of choice for long term management of diabetes. It was strongly proposed that even when drug treatment has begun, life-style changes and education remain an important part of managing diabetes ( Ipp E, 2000). While the majority of available synthetic anti-diabetic drugs target the dual metabolic defects that characterize T2DM, impaired insulin secretion and insulin resistance, some of these drugs (e.g., metformin) can have negative side effects at high doses (Ohmura et al.1998; Mudaliar and Henry 2001; Carroll et al, 2003; Fonseca, 2003). Thus, a major goal of antidiabetic research is the discovery of anti-hyperglycemic agents that are safe and that lack any negative side effects.

2.3.4 Side effects of drug and precautions

The most common side effects seen with rosiglitazone alone or in combination with metformin are upper respiratory tract infection, headache, back pain, hyperglycemia, fatigue, sinusitis, diarrhoea, and hypoglycemia. Rosiglitazone has been shown to cause mild to moderate accumulation of fluid (edema) and can lead to heart failure. Patients that already have heart failure may develop worsening symptoms with rosiglitazone. In addition, anemia may occur with rosiglitazone alone or combined with metformin. Rosiglitazone also causes increasing amounts of weight gain with increasing doses (Saltiel & Marks,

http://www.medicinenet.com/rosiglitazone/article.htm)

2.3.5 Traditional plant medicines as treatments for diabetes

More than 400 traditional plant treatments for diabetes mellitus have been recorded, but only a small number of these have received scientific and medical evaluation to assess their efficacy. Traditional treatments have mostly disappeared in occidental societies, but some are prescribed by practitioners of alternative medicine or taken by patients as supplements to conventional therapy. However, plant remedies are the mainstay of treatment in underdeveloped regions. A hypoglycemic action from some treatments has been confirmed in animal models and non-insulin-dependent diabetic patients, and various hypoglycemic compounds have been identified. A botanical substitute for insulin seems unlikely, but traditional treatments may provide valuable key or the development of new oral hypoglycemic agents and simple dietary adjuncts.

2.3.5.1 Herbal extracts as anti-diabetic

Herbal drugs are prescribed widely, even when their biologically active compounds are unknown, because of their effectiveness, lesser side-effects and relatively low cost (Valiathan, 1998). Many countries, especially in the developing world, have a long history of the use of herbal remedies in diabetes (McNeill,2006). The usual treatment is with concentrated (root) herbal extracts, commercialized 100 times cheaper than equivalent artificial drugs. The first registered use of anti-diabetic drugs was as herbal extracts used by Indians in the Amazon basin for the treatment of type 2 diabetes, and today promoted as vegetable insulin although not formally an insulin analog (Soumyanath, 2005). Phytochemical analysis of the herbal extracts are accumulating with most studies reported various kinds of flavanone glucosides (myrciacitrins) and acetophenone glucosides (myrciaphenones) with inhibitory activities on aldose reductase and -glucosidase (Matsuda, Morikawa, and Yoshikawa, 2002).

2.4 Hypertension
One of the long-term complications of diabetes is hypertension or high blood pressure. Hypertension and Type 2 diabetes are interrelated metabolic disorders (Sowers and Epstein 1995; Bakris et al. 2000) that strongly predispose an individual to atherosclerotic cardiovascular disease and to renal failure (Epstein & Sowers 1992). According to the World Health Organization, more than 1 billion individuals worldwide are hypertensive and 4 million individuals die annually as a direct result of hypertension. High blood pressure causes 1 in every 8 deaths, making hypertension the third leading killer in the world (Khatib. et. al 2005).

One of the most important intermediary factors for controlling hypertension is the action of the angiotensin converting enzyme-I (ACE-I) (Hernadez-Ledesma et al. 2003) which is an important enzyme involved in maintaining vascular tension. ACE activates a histidyl-leucine dipeptide called angiotensin I into a potent vasoconstrictor called angiotensin II (Skeggs et al. 1956). Angiotensin II also stimulates the synthesis and release of aldosterone, which increase blood pressure by promoting sodium retention in the distal tubules (Lieberman 1975). As such, the inhibition of ACE is considered a useful therapeutic approach in the treatment of high blood pressure in both diabetic and nondiabetic patients (Johnston and Franz 1992; Erdos and Skidgel ,1987). Recent studies indicate that flavonoid-rich food and plants have the ability to inhibit ACE-I activity, both in vitro and in vivo (Actis-Goreta et al. 2003; Kwon et al.2006). The consumption of selected flavonoid-rich foods may mimic synthetic ACE-I inhibitors and provide health benefits, but without adverse side effects. Phenolic phytochemicals are secondary metabolites of plant origin that constitute one of the most abundant groups of natural metabolites and form an important part of both human and animal diets (Bravo 1998; Crozier et al. 2000; Vattem et al. 2005). Recent studies have shown that phenolic phytochemicals have high antioxidant activity and certain therapeutic properties (Shetty et al. 2005) including anti-diabetic and anti-hypertension activity (Kwon et al. 2006). Inhibitors of angiotensin converting enzyme (ACE) are frequently used in therapy to reduce morbidity and mortality of patients with hypertension and other related diseases. ACE inhibition leads to a decrease in the level of the vasoconstricting peptide, angiotensin II, and a corresponding increase in the level of the vasodilatory peptide, bradykinin, therefore yielding an

overall reduction in blood pressure. Peptides with ACE-I activity have already been isolated from different food proteins (Ariyoshi, 1993; Yamamoto, 1997). An important determinant causing hypertension is the over activity of the renninangiotensin system, thereby making this a major target for therapy (Sarkissian, et al 2006). Renin which is protease synthesized by the kidneys, transforms angiotensinogen to angiotensin I, a biologically inactive decapeptide. ACE subsequently converts angiotensin I to an active octapeptide vasoconstrictor, angiotensin II, causing the contraction of blood vessels and thereby raising blood pressure (Lee et al, 2004). Antihypertensive drugs have been isolated from a number of plant species (Goretta L, et al 2005). It is now believed that screening plant extracts for inhibition of ACE will be an effective method to search for new anti-hypertensive agents (Wagner et al 1991). Various antihypertensive drugs were developed including captopril, ramipril, enalapril, cilazapril, pentopril, trandolapril, perindopril, lisinopril, alacepril, and imidaprilat (Suetsuna, 2004). Despite the advantageous effects of blocking the conversion of angiotensin I to angiotensin II or inhibiting the breakdown of bradykinin, these pharmaceutical products may have side effects such as cough, taste disturbances, skin rashes, and allergic reactions (Lee et al, 2004). For this reason, natural substances exhibiting ACE inhibitor activity interest scientists because of their safety, economy, and diversity. Apart from natural antihypertensive products derived from microbial sources (species of Doratomyces putredinis, Nocardia orientalis, Streptomycetes, Actinomycetes, and

Actinomadura) antihypertensive peptides isolated from various food protein sources including gelatin, tuna, bonito, corn, chickpea, mushroom, beef, mussels, buckwheat, garlic and soybean have been discovered. The peptides responsible for the anti-hypertensive activities were reported

as dipeptide (Val-Tyr) from sardines, tripeptide (Val-Pro-Pro, Ile-Pro-Pro) from sour milk, and dodecapeptide from casein (Ma et al 2006; Kuba et al, 2003 ).

2.5 Ocimum basilicum

Basil (Ocimum basilicum) of the family Lamiaceae is used in traditional medicine and as a culinary herb by virtue to its well-known source of flavouring principles (Javanmardi, 2003). This plant is a kind of low-growing herb that grows as a perennial in warm, tropical climates. Basil is originally native to India and other tropical regions of Asia. The variety of basil which is used in Italian food is typically called sweet basil, as opposed to Thai basil or holy basil, which is used in Asia.

2.5.1 Phytochemical components of O. basilicum

Many herb spices, especially those belonging to the Lamiaceae family, such as sage, oregano, and thyme, show strong antioxidant activity (Hirasa & Takemasa,1998). The genus Ocimum, a member of the Lamiaceae family, contains between 50 and 150 species of herbs and shrubs (Simon, Morales, Phippen, Vieira, & Hao 1999). A number of phenolic compounds with strong antioxidant activity have been identified in these plant extracts (Nakatani, 1997). The various basils have such different scents because the herb has a number of different essential oils which come together in different proportions for various breeds. The compound (E)-beta-caryophyllene (BCP) and -carotene found in basil is also found in oregano and could help to treat inflammatory bowel diseases and arthritis. It interacts selectively with one of two cannabinoid receptors, CB2, blocking the chemical signals that lead to inflammation without triggering cannabis's mood-altering effects. Herbs such as basil and oregano contain large amounts of these compounds (Gertsch et al, 2008).

2.5.2 Health benefits of O.basilicum

The potent antioxidant compounds in O. basilicum have been reported to exhibit antiaging, anti-cancer, anti-viral, and anti-microbial properties (Manosroi 2006). In addition, O. basilicum has also been shown to decrease the occurrence of platelet aggregation and experimental thrombus in rats (Tohti et al, 2006) and is widely used traditionally for supplementary treatment of stress, asthma and diabetes in India (Duke, 2008). The fasting and postprandial blood and urinary glucose levels in Type 2 diabetic patients were reduced after the

consumption of this herb with the added advantage of mild reduction of cholesterol level (Tiwari & Rao, 2002). Despite these health benefits O. basilicum like other aromatic plants such as fennel and tarragon, contains estragole, a known carcinogen and teratogen in rats and mice. While human effects are currently unstudied, studies in rodents indicate that it would take 1001000 times the normal anticipated exposure to become a cancer risk (Emea, 2004).

2.6 Mentha piperita

Mentha piperita (peppermint) is a hybrid mint, a cross between watermint (Mentha aquatica) and spearmint (Mentha spicata). It was first described as a species from specimens collected in England but is now universally agreed to be a hybrid (Harley,1975). Its pleasant taste makes it an excellent gastric stimulant (Budavari et al.1989). M. piperita has high menthol content, and is often used as a flavouring in tea, ice cream, confectionery, chewing gum, and

toothpaste. The oil also contains menthone and menthylesters. It is the oldest and most popular flavour of mint-flavoured confectionery (Gracindo, 2006).

2.6.1 Phytochemical content of M. piperita

M. piperita leaves contain oil that is high in menthol which act as a relaxant and releases tension from the body's digestive tract. In addition this herb reported contain ferulic acid (Sharma & Tyagi, 1991, Shasany et al. 2000). The most important compounds in peppermint are menthol, menthyl acetate, menthone, menthofuran, and pulegone (ESCOP, 1992) as well as limonene, eucalyptol, iso menthol, and isomenthone (European Pharmacopeias, 1994).

2.6.2 Health Benefits of M. piperita

M. piperita helps against upset stomachs, inhibit the growth of certain bacteria, and can help soothe and relax muscles when inhaled or applied to the skin. Other health benefits are attributed to the high manganese, vitamin C and vitamin A content, as well as trace amounts of various other nutrients such as fibre, iron, calcium, folate, potassium, tryptophan, magnesium, omega-3 fatty acids, riboflavin, and copper (Gracindo, 2006). M. piperita relaxes the gastroesophageal sphincter, thus promoting belching. M. piperita is digestive, carminative, antispasmodic, cooling, and is a mild stimulant (Salem, 1995). It is commonly used for digestive problems such as cramps, bloating, gas, nausea, loss of appetite, and irritable bowel syndrome. It can also be used as a painkiller to treat toothaches, headaches, and migraines. It relieves burning, can be applied to congested skin, relieves the pain of sprains and strains, and helps fight bad breath. M. piperita is also very good for treating coughs, colds, and fevers (Salem, 1995).

2.7 Anethum graveolence

Anethum graveolens known as dill, is an annual herb growing in the Mediterranean region, Europe, central, southern Asia and it is widely cultured in south eastern region of Iran. The plant is used both medicinally and as an aromatic herb and spice and cookery. A. graveolence is a short-lived annual herb. It is the sole species of the genuse Anethum, though classified by some botanists in a related genuse as Peucedanum graveolens (L.) C.B.Clarke. (Zohary, et al 2000). A. graveolence is said to be best when used fresh, as it loses its flavour rapidly if dried. However, freeze-dried dill leaves preserve their flavour relatively well for a few months. A. graveolence seed is used as a spice with a flavour similar to caraway. A. graveolence oil can be extracted from the leaves, stems and seeds of the plant (Zohary, et al 2000).

2.7.1 Phytochemicals in A. graveolence

A. graveolence contains phytochemical such as tannin, flavonoids and steroid. The seeds of A. graveolence contain the essential oil carvone. A. graveolence also produces petroselenic fatty acids. The presence of flavonoids, phenolic compounds and essential oil in A. graveolens

has been reported (Ishikawa T, et al. 2002) Some pharmacological effects of the plant such as antimicrobial (Chaurasia and Jain, 1978) antispasmodic (Fleming T, 2000) anti secretary and mucosal protective effects have also been reported (Hosseinzadeh et al 2002) . The anti-hyper cholesterolaemic and anti-hyperlipidaemic activities (TC, TG) of the crude extract have previously been reported (Yazdanparast and Alavi, 2001).

2.7. 2 Medicinal Uses

A. graveolence has been used traditionally for gastrointestinal ailments such as flatulence, indigestion, stomachache colic and to tract intestinal gas (Hosseinzadeh et al, 2002). A. graveolence is used clinically as an antispasmodic, carminative, diuretic, stimulant for lactation (De Rougemont et al, 1989). A. graveolence also has calcium, which acts as an anti-bacterial agent and offers protection from carcinogens and free radicals. A. graveolence is known medicinally for its sooting effects on distressed stomach and is safe for children and adults. Many herbalists recommend combining A. graveolence and fennel to relieve colic in infants (Yazdanparast et al., 2001).

3.1 Materials

3.1.1 Herbs
The three herbs (Mentha pipperita, Anethum gravolence and Ocimum basilicum) used in the present studies were purchased in dried form from local shops in Iran and herbs were dried to constant weight and ground to pass through 1 mm screen. All ground herbs were and were kept in clean and dry air tight dark glass bottle and the bottle were placed at room temperature away from direct sunlight.

3.1.2 Starter Culture

Yogurt-Mix starter culture used contains the mixture of the following bacteria:

Lactobacillus acidophilus LA-5, Bifidobacterium Bb-12, Lactobacillus casei LC-01 and Streptoccus thermophilus Th-4 in the ratio of 4:4:1:1 All chemicals were of analytical grade (A.R.) and were purchased from Sigma.

3.2 Methods
3.2.1 Water extraction of herbs
The ground herbs were suspended in distilled water (dH2O) in the ratio 1:10. The mixture was left to incubate overnight in a water bath (70C) followed by centifugation (15 minutes, 2000 rpm at 4C). The supernatant (water herbal extract) was used in the experiments.

3.2.2 Yogurt
3.2.2.1 Preparation of starter culture

Pasteurized full-cream fresh cows milk (1 litre) was warmed (41C) and a small volume (100ml) of the warm milk was placed into a clean and sterilized container. Yogurt-mix starter culture was added into the milk. The starter mixture was stirred carefully and the microbial suspension was then mixed thoroughly with the remainder of the milk. Incubation was carried out at 41C overnight. The yogurt formed was kept at 4C and used as starter culture within a week.
3.2.2.2 Preparation of yogurt

Full cream ( 4% Fat ) milk was used throughout the present studies. Water herbal extract (10ml) was added into 85 ml of pre-heated full cream milk (10% Total Solid). This was followed by the addition of 2 g full cream milk powder (10% Total Solid) (to correct the milk solid content) and 5 ml of starter culture. The mixture was stirred carefully and incubation was carried out at 41C. The same process was carried out for plain yogurt preparation except that 10 ml of dH2O was added in place of water herbal extract. All yogurts were incubated at 41C in the same incubator and stored at 4C when the pH was reduced to 4.5.

3.2.3 pH and TTA

The changes of pH during fermentation of yogurts were monitored every half an hour until the pH reached 4.5. Sample of yogurts (3ml) was mixed with 3 ml of dH2O for pH measurement ( Mettler-Toledo 320). The total titratable acid (TTA) in yogurt was determined by titration using 0.1N NaOH. Yogurt sample (1ml) was transferred into an

Erlenmeyer flask containing 9 ml dH2O. Several (3-5) drops of 0.1% phenolphthalein as pH indicator were added. NaOH (0.1 N) was titrated into the solution and the solution was mixed thoroughly. The process was repeated until the indicator changed to a constant pink colour. Since 1 ml of 0.1 N NaOH is neutralized by 0.009g of acid, the amount of acid produced during fermentation can be calculated as follows:
Percentage of Lactic Acid=Dilution factor (10) * V NaOH *0.1N* 0.009 * 100%

3.2.4 Preparation of yogurts extracts

Plain and herbal (10g) yogurts were homogenized with 2.5 ml of sterile distilled water. The pH of the yogurts was determined and the yogurts subsequently acidified to pH 4.0 with HCl (0.1M). The yogurts were then heated in water bath (45C) for 10 minutes followed by centrifugation (5000g, 10 minutes 4C). NaOH (0.1M) was added to adjust the pH of supernatant to pH 7.0. The supernatants were centrifuged again at (5000g, 10 minutes 4C) and the supernatant was harvested and stored in a -20C freezer until required for analysis.

3.2.5 Antioxidant activity determination by 1,1-Diphenyl-2-Picrylhydrazyl (DPPH) radical inhibition assay (Apostolidis, et al. 2006)
Each homogenized water extract (250l) was added in to 3 ml of 60 mM DPPH in ethanol. The decrease in absorbance was monitored at 517 nm until a constant reading was obtained. The readings were compared with the controls, which contained 250 l of dH2O instead of the extract. The % inhibition was calculated as follows:

A517 control A517 extract

% inhibition= [A517 control]

x 100

3.2.6 Total Phenolic assay

The total phenolic content was determined by an assay modified by Shetty et al (1995). Briefly, 1 ml of water yogurt extract was transferred into a test tube and mixed with 1 ml of 95 % ethanol and 5 ml of dH2O. Folin-Ciocalteu reagent (50% v/v; 0.5 ml) was added to each sample followed by thorough mixing using a vortexer. After 5 minutes 1 ml of 5% Na2CO3 was added to the reaction mixture and these were left to stand for 60 minutes at room temperature. The absorbance was read at 725 nm. The absorbance values were converted to total phenolics and were expressed in microgram equivalents of gallic acid per millilitre (ml) of the sample. Standard curves were established using various concentration of gallic acid (5-60 g/ml) in Methanol (Figure 3.1).

1.4 1.2 1 Abs (725nm) 0.8 Gallic acid 0.6 0.4 0.2 0 0 10 20 30 40 50 60 70 y = 0.0197x + 0.0149 R = 0.9966

Weight (g/ml)

Figure 3.1: typical standard curve of gallic acid for the estimation of total phenolic in yogurts.

3.2.7 O-Pthaldialdehyde Assay (OPA)

Protein content in the yogurts water extract was determined using the OPA method as described by Goodno et al (1981).
3.2.7.1 Preparation of OPA reagent

The OPA solution was made by combining the following reagents: 25 ml of 100 mM sodium tetraborate, 2.5 ml of 20% (wt/wt) sodium dodecyl-sulphate (SDS), OPA (40mg dissolved in 1 ml of methanol) and 100 l of -mercaptoethanol. The final volume was made up to 50 ml using dH2O. OPA reagent is light-sensitive and thus must be protected from light sources during preparation and during the assay. This reagent was prepared fresh and used within 2 hours of preparation.

3.2.7.2 OPA assay

To assay proteolysis with milk protein as substrates, a small aliquot of yogurt extract (usually 10 to 50 l containing 5 to 100 g proteins) was added directly to 1.0 ml of OPA reagent in a 1.5 ml cuvette. The solution was mixed briefly by inversion and incubated for 2 minutes at room temperature. The absorbance reading was taken at 340 nm (Spectronic 10 UV (190-1100 nm)) model: Genesys made in USA). The peptide concentration was estimated against the tryptone standard curve (see section 3.2.7.3).
3.2.7.3 Tryptone standard curve

A standard curve of peptide concentration was prepared using trypton. Various concentrations of trypton standards (0.25, 0.5, 0.75, 1.00, 1.25, 1.50 mg/ml) were prepared from the stock solution. The trypton standards were prepared and treated in the same manner for samples for each OPA assays. The resulting liner regression between abs (340nm) and trypton

standard (mg/g) was constructed and used in the estimation of OPA peptides in sample.
0.9 0.8 0.7 0.6 abs(340nm) 0.5 0.4 0.3 0.2 0.1 0 0 0.2 0.4 0.6 0.8 weight(mg/g) 1 1.2 1.4 1.6 y = 0.5416x + 0.0336 R = 0.9927

Figure3.2: Typical Standard curve of tryptone for OPA peptides estimation

3.2.8 ACE inhibition assay (Vermeirssen et al. 2002)

3.2.8.1 Preparation of rabbit lung extract

Fresh rabbit lung were snap-frozen to -20C followed by grinding using pestle and mortar and subsequently homogenized with ice-cold 0.05 M potassium phosphate buffer, pH 8.3
(Vermeirssen et al. 2002) . The homogenized rabbit lung was then centrifuged for 60 minutes at

20000 g. The clear red supernatant possessed high ACE activity and was aliquoted into ampoules of 1.0 ml. The aliquots were stored in -20C freezer until required for analysis.

3.2.8.2 Preparation of standard rabbit lung

The standard rabbit lung was prepared as follow: 2.5 ml of deionized water was added to standard rabbit lung extract (0.25 U/ml; Sigma.Co) to obtain a final concentration of 0.1 U/ml. The solution was then aliquoted into ampoules of 500 l and stored in -20C freezer until required for ACE inhibition assays.
3.2.8.3 Preparation of ACE reagent

The ACE reagent was prepared as described by Vermeirssen et al. (2002). Sodium chloride (NaCl, 2.34 g) was dissolved in 80 ml of dH2O and the volume was made up to 100 ml in a volumetric flask. Solution A was made by mixing 0.607 g of Tris in 50 ml of dH2O. The pH was adjusted to 8.3 and the volume made up to 100 ml. Furanacryloyl-Phe-Gly-Gly (FAPGG: 25 mg) was mixed in 62.6 ml of solution A. The dissolved FAPGG (ACE reagent) was subsequently aliquoted (500 l) into ampoules and these were stored in -20C freezer until required for analysis.
3.2.8.4 Preparation of Enalapril

The inhibition solution, enalapril was prepared according to Vermeissen (2002). The 103

M stock solution was prepared in demineralised water and stored at -20C. For the alkaline

hydrolysis of enalapril to enalaprilat, 4 ml of 1M NaOH was added to 10 ml of 10-3 M enalapril stock solution in demineralised water. The reaction mixture was then placed at room temperature overnight. On the next day, the pH of the mixture is adjusted to pH 8.0 using 1M HCI followed by diluting the solution to 20 ml with demineralised water. The final concentration of analaprilate, 10-4M was then aliqouted into ampoules of 500 l. The aliquotes were stored in 20C freezer until required analysis.

In order to validate the ACE assay, 9 different concentrations of enalapril were prepared by serial dilution from the 10-4 M stock solution to yield 10-5, 10-6, 10-7, 10-8, 10-9, 10-10, 10-11 and 10-12 M. This was carried out as follows: Stock solution (0.1 ml: 10-4 M) was added into 0.9 ml of sterile distilled water in a test tube to yield 10-5 M of enalapril. The mixture was mixed thoroughly and the process was repeated using fresh sterile distilled water to dilute until enalapril was diluted to 10-12 M.

3.2.8.5 Measurement of rabbit lung extracts ACE activity (control)

The method used to determine the ACE activity was adapted from Vermeirssen et al. (2002). ACE reagent (500 l) was added to 300 l of dH2O (blank) in a cuvette. The mixture was mixed thoroughly and incubated in a water bath (37C) for 2 minutes. The mixture was then taken out from the water bath and 300 l of rabbit lung extract was added and the mixture was mixed thoroughly. The enzymatic reaction was allowed to take place in the water bath (37C) for a total period of 20 minutes. The cuvette was taken out briefly every 5 minutes for the measurement of absorbance reading at 340 nm.

3.2.8.6 Measurement of standard rabbit lung (Sigma) ACE activity

ACE reagent (500 l) was added to 500 l of dH2O (blank) in a cuvette. The incubation was carried out as described in section 3.2.7.5, except that 100 l of standard rabbit lung enzyme (Sigma Co.) was used.

3.2.8.7 Measurement of rabbit lung extract ACE activity in the presence if inhibitor (Enalapril)

Enalapril was used as the reference compound in the assay. The assay was carried out as described in section 3.2.7.5, except that 300 l of dH2O was replaced by 300 l eanalpril (10-4 to 10-12 M).

3.2.8.8 Measurement of anti-ACE activities of water yogurt extract

The assay was carried out as described in section 3.2.7.5, except that 300 l of water extract prepared from yogurt at different storage days was used in place of the 300 l of dH2O.

3.2.8.9 Calculation of ACE inhibition activity

ACE activity (unit/min) was calculated by dividing the difference in absorbance (unit) between the 5th and 20th minute of incubation by 15 minute: ACE activity (unit/min) = (Abs 20' - 5') /15 min The rate of the reduction in ACE inhibition activity was calculated as follow: If in comparison to control ACE activity (blank)
ACE inhibitory = activity (%) ACE activity (control) ACE activity (yogurt extract) X 100 ACE activity (control)

Specific activity was calculated as below: Specific activity = ACE activity / [peptide] in 300 l (unit/min/mg protein)

3.2.9 -Amylase inhibition assay (Apostolidis et al. 2007)

3.2.9.1 -Amylase enzyme solution

Porcine pancreatic -amylase (EC 3.2.1.1) was purchased from Sigma Chemical Co.. According to the manufacture product description, one unit of enzyme will liberate 1.0 mg of maltose from starch per minute at pH 7.0 at 20C. The enzyme concentration used in assay was 0.5 mg/ml (Apostolidis et al. 2007). Enzyme powder was dissolved in pre-chilled 0.02 M sodium phosphates buffer, pH 6.9 with 0.006 M sodium chloride, yielding a clear to hazy solution. This is due to the pressure of enzyme carriers, lactose which are partially soluble in the chilled buffer. The enzyme solution was prepared freshly and stored at temperature below 4C prior to assay.

3.2.9.2 Sodium phosphate buffer (0.02 M), pH 6.9 with .006 M sodium chloride

The following three solutions were prepared separately. 200 ml dH2O was added to 1.582 of Na2HPO4 200 ml dH2O was added to 1.062 of Na2PO4 100 ml dH2O was added to 0.3506 g of NaCl All 3 solutions were then mixed well followed by the addition of 400 ml dH2O as to obtain the desirable pH of 6.90. If the pH deviates from 6.9, the pH was adjusted by adding either Na2HPO4 as base or NaH2PO4 as acid. Finally the solution was brought up to the final volume of 1000 ml in volumetric flask. The buffer prepared was stored at 25C and used within 2 weeks.

3.2.9.3 Dinitrosalicyclic acid (DNSA) reagent

The original DNSA reagent developed by Summer and Sisler (1944) contained 0.63% DNSA, 18% titrate, 0.5% phenol, 0.5% sodium bisulfite, and 14% NaOH. A modified DNSA reagent used in this study reagent was prepared by dissolving (constant stirring) 2% (w/v) of DNSA with 1% NaOH. The other component of the modified DNSA reagent, the 18.2 % (w/v) potassium sodium tartrate, also known as Rochelle salts, was prepared separately using dH2O. The reagent was prepared freshly prior to assay. Precautionary steps were taken as carbon dioxide tends to interfere the stability of reagent. In addition this colour reagent should be protected from all light sources.
3.2.9.4 1% starch solution

Soluble starch (1g) was dissolved in 100 ml of sodium phosphate buffer. Constant stirring at 90C helped the dissolution of starch in buffer. The starch solution was then cooled and stored at 4C. The starch solution was incubated at 25C for 5 minutes prior to assay.

3.2.9.5 -Amylase inhibition assay

The -Amylase inhibition assay was adapted from Shetty et al. (2006). Yogurt extracts (500l) were mixed in 500l of 0.02 M sodium phosphate buffer, pH 6.9 with 0.006 M sodium chloride containing 0.5 mg/ml. The -amylase solution was incubated at 25C for 10 minutes. After pre-incubation, 500 l of a 1% starch solution in 0.02 M sodium phosphate buffer, pH 6.9 with 0.06 M sodium chloride was added to each tube at predetermined time intervals. The reaction mixtures were then incubated at 25C for 10 minutes. The reaction was stopped with using 1.0 ml of dinitrosalicyclic acid (DNSA) colour reagent. The test tubes were then incubated in a boiling water bath for 7 minutes. Subsequently, 1.0 ml of 18.2 % tartrate solution was added

to the each tube after the boiling session, before cooling to room temperature. The reaction mixture was then diluted after adding 10 ml of dH2O. Absorbance was measured at 540 nm. The formula used to calculate enzyme inhibition is stated as follows: Inhibition percentage = (Absorbance of control Absorbance of extract) Absorbance of control *100

3.2.10 -Glucosidase inhibition (Apostolidis et al. 2007)

3.2.10 .1 -Glucosidase enzyme solution

1000 U of alpha-glucosidase was dissolved well in 3960l (3.96 ml) of 0.1M potassium phosphate buffer (pH 6.90) and aliquoted into 33 ampoules and stored at -20C. Each ampoule contained 30U/120l of alpha-glucosidase enzyme solution.
3.2.10.2 0.1M potassium phosphate buffer (pH 6.90)

The following two solutions were prepared separately: 200 ml dH2O was added to 9.11 g K2HPO4 200 ml dH2O was added to 6.49 g KH2PO4 Both 2 solutions were then mixed well followed by the addition of 400ml dH2O as to obtain the desirable pH of 6.90. If the pH deviates from 6.90, the pH was adjusted by adding either K2HPO4 as base or KH2PO4 as acid. Finally the solution was brought up to a final volume of 1000 ml in volumetric flask. The buffer prepared was stored at 25C and used within 2 weeks.
3.2.10.3 5mM p-nitrophenyl--D-glucopyranoside substrate solution

Potassium phosphate buffer (0.1M,pH 6.90) was slowly added into 5mM p-nitrophenyl--Dglucopyranoside until it fully dissolved. This solution was freshly prepared prior to running glucosidase assay.

3.2.10.4 -Glucosidase Inhibition Assay

The alpha-glucosidase inhibition assay was performed essentially as described by Apostolidis et al (2006) with some modifications. The reaction mixture contained 500l of sample extract and 1ml of 0.1M potassium phosphate buffer (pH 6.90) containing -glucosidase solution (1.0 U/ml) and incubated in water bath at 25C for 10 minutes. After 10 minutes 500 l of 5 mM p-nitrophenyl--D-glucopyranoside solution in 0.1 M potassium phosphate buffer (pH 6.9) was added to each tube timed intervals. The reaction mixture was further incubated at 25C for 5 minutes. Absorbance reading was measured spectrophotometrically at 405 nm, before and after incubation period. The reading was compared to a control which had 500 l of buffer solution in place of the extract. The -glucosidase inhibitory activity was expressed as inhibition % as follows:
Inhibition percentage = (Absorbance of control Absorbance of extract) Absorbance of control * 100

3.2.11 Calculation of IC50 for enzyme inhibition activity Enzyme inhibition was expressed as the concentration of inhibitory compound that inhibition 50 % of ACE activity (IC50), assuming that the activity of the blank is 100%. For ACE inhibition studies 3 different strength of yogurt extracts (300 l, 150 l, 75 l; dH2O was used to make up the volume to 300l) were determined. For amylase and glucosidase inhibition study; 3 different strength of yogurt extracts (500l, 250l and 125l; dH2O was used to make up the

volume to 500l) were determined. IC50 values were calculated by subjecting the data to a nonlinear adjustment programme (PRISM version 4.02 for Windows (GraphPad Software, Inc. San Diego, CA, USA)) which estimates the value of the IC50 together with the standard error. 3.2.12 Statistical analysis Three independent batches of yoghurts were prepared. Each batch consists of yogurts stored for 0, 7, 14, 21 and 28 days. All samples were analysed in duplicate. Data were analyzed using one-way ANOVA by SPSS, version 12.0 and t-Test by Microsoft Excel 2003. Duncans multiple range tests was used to compare the means among the days when a significant variation was established by ANOVA at p 0.05.

4.1 Change of yogurts pH

pH of yogurts was measured during fermentation until a constant reading was obtained (Figure 4.1). No differences in initial pH were recorded for all yogurts. There was a lag phase of about 60 minute at the beginning of the incubation before the pH of yogurts began to reduce. The pH of plain yogurt began to reduce significantly (p<0.05) during minute 120 and 240. The addition of herbs into yogurt was shown to reduce pH at a faster rate than that in plain yogurt. All herbal yogurts showed lower pH readings (pH 5.4-5.7) than plain-yogurt (pH 6.12) at the 120th minute of incubation. pH 4.5 was achieved fastest for A. graveolenceyogurt followed by O. basilicum-,

M. piperita- and plain-yogurts (240, 300, 300 and 420 minute respectively). All herbal yogurts reached constant pH at 360th minute whereas plain-yogurt reached constant pH at the 420th minute of incubation.
10.00 9.00 8.00 7.00 6.00 pH 5.00 4.00 3.00 2.00 1.00 0.00 0 30 60 90 120 150 180 210 240 270 300 330 360 390 420 Time(min) plain O. basilicum M. piperita A. graveolence

Figure 4.1. Changes of pH of yogurts during lactic acid bacteria fermentation of milk in the presence of herbs at 41C

4.2 Total titratable acid (TTA) of yogurts

Yogurt TTA, which measure the equivalent percentage (%) of lactic acid present in the yogurt during fermentation, is as shown in Figure 4.2. The increase in TTA for the plain yogurt was almost linear in comparison to sigmoidal curve for pH changes during incubation. The TTA for the three herbal-yogurts were higher than plain yogurt at all time points during the incubation. Both O. basilicum and M. piperita-yogurts had the same rate of TTA production, whereas A. graveolence-yogurt had a higher rate of TTA production during the fermentation between the 180th- 240th minute of fermentation.

0.90 0.80 0.70 TTA(% Lactic Acid) 0.60 0.50 0.40 0.30 0.20 0.10 0.00 0 30 60 90 120 150 180 210 240 270 300 330 360 390 420 Time(min)
plain O. basilicum M. piperita A. graveolence

Figure 4.2. Changes of total titratable acid (TTA) in yogurt during lactic acid bacteria fermentation of milk in the absence or presence of herbs at 41C.

4.3 Total phenolic content (TPC) in yogurts

Herbs contain phenolic compounds. Mixing of herbs with milk resulted in higher TPC for M. piperita- and A. graveolance-milk mixtures. Fermentation resulted in increased TPC in all herbal-yogurts. Highest total phenolic content was recorded in A. graveolence-yogurt (35g/ml) followed by M. piperita-yogurt, O. basilicum-yogurt and plain-yogurt (26g/ml, 20g/ml, and 15g/ml respectively).

60 total phenolic content(g/ml) 50 40 30 20 10 0 BF 0 7 days 14 21 28 plain O. basilicum M. piperita A. graveolence

Figure 4.3: total phenolic content of water extract of milk + herbs before fermentation, fresh yogurts (0
day) and yogurts stored (4C) up to 28 days.

The TPC increased gradually at the same rate during the first week and the last two weeks of storage for all herbal-yogurts whereas plain-yogurt showed constant smaller increase in TPC during the 28th day of storage at 4C. Maximum TPC 47.68, 37.51, 28.56, and 19.51g/ml for A. graveolence-, M. piperita-, O. basilicum- and plain-yogurts respectively were recorded on day 28 of storage.

4.4 Antioxidant capacity of yogurts

Antioxidant activities of mixture of herbs and milk showed higher (p<0.05) for A. graveolance and M. piperita) than milk alone. Plain-yogurt showed the lowest antioxidant capacity throughout the storage period, with maximum capacity recorded on day 7 of storage. Both A. graveolance- and M. piperita-yogurts had higher (p<0.05) antioxidant activity than that in plain yogurt, both at the end of fermentation (fresh yogurt) and throughout the storage period.

Compared to initial values, only M. piperita-yogurt showed higher (p<0.05) antioxidant value as a result of fermentation. Highest antioxidant activity on day 7 was recorded by A. graveolence yogurt (59%) followed by M. piperita- O. basilicum- and plain-yogurts (52, 45 and 33% respectively), but only O. basilicum-yogurt showed significantly (p<0.05) higher antioxidant values (45 4.35 %) than its fresh yogurt value (35 1.37 %).
70 60 DPPH Inhibition(%) 50 40 30 20 10 0 BF 0 7 days 14 21 28
plain O. basilicum M. piperita A. graveolence

Figure 4.4: Inhibition (%) of DPPH scavenging activity by water extract of milk + herbs before fermentation, fresh yogurts (0 day) and yogurts stored (4C) up to 28 days.

Continued storage at 4C longer than 14 days resulted in reduction in antioxidant activities for all yogurts. Highest percentage of reduction in antioxidant activity between day 7 and day 28 of storage was showed in O.basilicum-yogurt (28%) followed by A.graveolence- , M.peppermint- and plain-yogurts (23, 21, and 17% respectively).

4.5 Concentration of OPA peptides

Except for plain-yogurt, OPA peptides in herbal-yogurts were slightly higher than their respective initial herb plus milk values (Figure 4.5). There was only a small increase in OPA peptides in plain-yogurt on day 7 compared to fresh yogurt, which remain relatively unchanged over the next 2 weeks. OPA peptides in plain-yogurt on day 28 of storage was lower (p<0.05) , (13.3mg/g) than that in fresh-yogurt (15.4mg/g). The OPA peptides in herbal-yogurts on the 7th day of storage increased to 28-36mg/g. The increase was highest for M. piperita-yogurt (33.4 mg/g) followed by A. graveolence- and O. basilicum-yogurts (31.5 and 30.2 mg/g respectively). The OPA peptides on day 28 was also higher (p<0.05) in M. piperita-, A. graveolence-, and O. basilicum-yogurts (26.75, 23.46, and 20.14 mg/g respectively) compared to plain-yogurt (13.32 mg/g).
40 35 30 peptide(mg/g) 25 20 15 10 5 0 BF 0 7 days 14 21 28
plain O. basilicum M. piperita A. graveolence

Figure 4.5. OPA peptides in water extract of milk + herbs before fermentation, fresh yogurts (0 day) and yogurts stored (4C) up to 28 days.

4.6 Angiotensin-1 converting enzyme (ACE)

The inhibition of rabbit lung ACE activity by yogurt water extracts is as shown in Figure 4.6. The inhibition of ACE by plain yogurt extract increased to 62.0% by day 7 of storage. The capacity to inhibit ACE activity reduces gradually thereafter to reach the lowest level (28.8 %) on day 28 of storage.

120 100 80 %inhibition 60 40 20 0 0 7 14 days 21 28 plain M. piperita O. basilicum A. graveolence

Figure 4.6. Effect of refrigerated storage (4C) of yogurt water extracts for 28 days on angiotensin-1
converting enzyme (ACE) inhibition (%).

All herbal yogurts showed anti-ACE activity higher than plain yogurt at respective storage period. M. piperita-yogurt had the highest inhibition on ACE activity throughout the

storage period (84.0, 91.0, 87.3, 74.0 and 63.6% on day 0, 7, 14, 21 and 28 respectively). A. graveolence-yogurt which had similar anti-ACE activity with O.basilicum-yogurt, showed higher (p<0.05) anti-ACE activity than plain yogurt at all storage period except on day 7 of storage. IC50 of ACE inhibition i.e. the concentration of substance needed to inhibit 50% of the original ACE activity were lower (p<0.05) for all herbal-yogurts than that for plain-yogurt throughout the storage period. For all yogurts; lowest IC50 were seen on days 7 and 14 of storage whereas highest IC50 values were observed on day 28 of storage. For each storage day, M. piperita-yogurt showed the lowest IC50 followed by A. graveolence- O. basilicum- and plainyogurts.
600 500 400 300 200 100 0 BF 0 7 days 14 21 28 plain M.piperita A.graveolence O.basilicum

Figure 4.7: Angiotensin converting enzyme IC50 of water extract of milk + herbs before fermentation, fresh yogurts (0 day) and yogurts stored (4C) up to 28 days.

IC50(mg/g)

4.7 Alpha-amylase inhibition

The inhibition of -amylase activity by yogurt extracts is as shown in Figure 4. 8. Plain yogurt on day 0 of storage inhibited 16.9% of -amylase activity. The inhibition of -amylase increased to 22.9% by day 7 of storage after which the value decreased gradually to the lowest (10.5%) on day 28 of storage. A. graveolence- and M. piperita-yogurts had higher -amylase inhibition (47.2 and 31.0% respectively) on day 0 of storage than that for control (16.8 %). Both herbal-yogurts showed higher (p<0.05) -amylase inhibition on day 7 (57.8 and 40.0% respectively) compared to that shown on day 0 (47.2 and 31.0% respectively). O.basilicum-yogurt inhibition of amylase (18.3%) was not different from plain yogurt (16.8%) on day 0. However its inhibition on -amylase was increased by almost 100% on day 7 (36.9%) of storage. All three yogurts showed gradual decrease in -amylase inhibition from day 7 to day 28 of storage.

70 60 50 inhibtion(%) 40 30 20 10 0 0 7 14 days 21 28

plain M. piperita O. basilicum A. graveolence

Figure 4.8. Effect of refrigerated storage (4C) of yogurt water extracts for 28 days on -amylase
inhibition (%).

The potency (IC50) of -amylase in all yogurts increased after 7 day of storage at 4C which was maintained until the 14th day of storage after which the potency decreased gradually toward the end of the storage period (day 28). The most potent effect on -amylase inhibition on day 7 of storage was shown by A. graveolence-yogurt (26.6mg/g) followed by M.piperita- and O.basilicum yogurts (37.2 and 42.0 mg/g respectively). All herbal-yogurts except, A.graveolence-yogurt, showed similar changes in potency (IC50) during storage to that shown by plain-yogurt. A.graveolence-yogurt had the highest potency on -amylase inhibition and the least changes in potency during the storage period.
140 120 100 IC50(mg/g) 80 60 40 20 0 BF 0 7 days 14 21 28 plain M. piperita A. gravelonce O. basillicim

Figure 4.9. -amylase IC50 of water extract of milk + herbs before fermentation, fresh yogurts (0 day)
and yogurts stored (4C) up to 28 days.

4.8 Alpha- glucosidase inhibition

The -glucosidase inhibition (%) by yogurt extracts are as shown in Figure 4.10.

45 40 35 30 % inhibition 25 20 15 10 5 0 0 7 14 days 21 28 plain O. basilicum M. piperita A. graveolence

Figure 4.10. Effect of refrigerated storage (4C) of yogurt water extracts for 28 days on glucosidase inhibition (%).

All yogurts showed maximal inhibition on -glucosidase on day 7 of storage (Figure 4.11) followed by gradual reduction in inhibitory activity thereafter. The inhibition of -

glucosidase for plain-yogurt on day 0 was 7.0% whereas that on day 7 was 14.4%. This inhibition reduced gradually after day 7 of storage to reach 4.4 % on day 28 of storage. Highest inhibition of -glucosidase on day 7 was shown by A. graveolence-yogurt (39.0%) followed by M. piperita-yogurt (22.6 %) and O. basilicum-yogurt (18.4%). M. piperita-yogurt on day 14 (21.0%) and day 21 (19.6%) maintained the highest (p<0.05) -glucosidase inhibition activity than that on day 0 (14.4%), whereas A. graveolence-yogurt sustained the highest -glucosidase inhibition from day 0 until day 28 of storage. The potency (IC50) of -glucosidase in all yogurts increased after 7 day of storage at 4C which was maintained until the 14th day of storage after which the potency decreased gradually toward the end of the storage period (day 28). A. graveolence-yogurt (26.6mg/g) showed the most potent effect on -glucosidase inhibition on day 7 of storage followed by M. piperita- and O. basilicum yogurts (37.2 and 42.0 mg/g respectively). With regard to changes in IC50, both plain- and O. basilicum-yogurts showed the greatest increase in potency during the first 7 days of storage. Plain and O. basilicum-yogurts also lost potency during storage but their IC50 values (348 and 208 mg/g respectively) on day 28 were still lower than their respective fresh yogurt values (365 and 246 mg/g respectively).

450 400 350 IC50(mg/g) 300 250 200 150 100 50 0 BF 0 7 days 14 21 28 plain O. basilicum M. piperita A. graveolence

Figure 4.11. -glucosidase IC50 of water extract of milk + herbs before fermentation, fresh yogurts (0 day) and yogurts stored (4C) up to 28 days.

4.9 Regression values of correlation between TPC and anti-diabetic enzyme inhibition activities, and OPA peptides and ACE activity
Table 4.1 Regression values of correlation between TPC and anti-diabetic enzyme inhibition activities, and OPA peptides and ACE activity Yogurt TPC vs amylase TPC vs -glucosidase TPC vs ACE OPA vs amylase OPA vs -glucosidase OPA vs ACE plain 0.47 0.25 0.05 0.01 0.06 0.04 M. piperita 0.63 0.32 0.18 0.12 0.18 0.49 A. gravoeolence 0.55 0.27 0.08 0.13 0.2 0.56 O. basilicum 0.44 0.27 0.13 0.07 0.14 0.29

Linear regression is used to describe the relationship between two quantitative variables (x and y). Correlation was found to exist between TPC and -amylase or -glucosidase but not between TPC and ACE. For all 4 yogurts, the correlation between TPC and -amylase was found to be stronger (r2 between 0.44 and 0.63) than between TPC and -glucosidase (r2 between 0.27 and 0.32). OPA peptides on the other hand was found to be moderate correlated with ACE (r2 between 0.04 and 0.56), but not with -amylase or -glucosidase (r2 between 0.06 and 0.2). For ACE, plain-yogurt showed poor correlation (r2= 0.044) whereas herbal-yogurt showed strong correlation (r2=0.49, r2=0.56, r2=0.29 for M. piperita-, A. graveolence- and O. basilicum-yogurts respectively.

5.1 Effect of herbs (M. piperita, O. basilicum and A. graveolence) on yogurt fermentation
Fermentation of milk occurred as a result of microbial growth, which utilizes lactose as its metabolic fuel. This is because the microbe will catabolise carbon sources (i.e. glucose and galactose) to yield energy under anaerobic fermentation to produce lactic acid. This results in the formation of lactic acid under anaerobic condition that reduces the pH of milk. Yogurt fermentation using conventional starter culture consists of two stages. In the first stage, L. bulgaricus proteolytic activity produces amino acids and small peptides that stimulate S. thermophilus growth (Sandine & Elliker, 1970) whereas S. thermophilus metabolites, carbon dioxide and formic acid, stimulate the growth of L. bulgaricus (Kailasapathy, 2006). Thus both S. thermophilus and L. bulgaricus have symbiotic growth and these bacteria exhibit a typical indirect positive interaction called proto-cooperation. At the end of the first stage, the growth of

S. thermophilus slowed down because of the high lactic acid concentration. This marks the beginning of the second stage, i.e. the slowing of pH reduction that eventually stabilized. The present studies used starter culture devoid of L. bulgaricus because this bacteria tends to increase acid production during refrigerated storage (Beshkova et al.,1998), which is not desirable from consumer point of view. Thus, in the present studies S. thermophilus which is also a proteolytic bacteria (Tamine & Deeth, 1980), play important role in providing necessary supply of peptides and amino acids for other microflora (Lactobacillus sp.) in the starter culture. pH value is used to measure the concentration of H+ contributed by the production of lactic acid by lactic acid bacteria (LAB). Thus, the differences in the rate of pH reduction may be attributed to the different growth rates of the probiotics in the yogurt which produce lactic acid. The microflora counts which were not evaluated in the present studies need to be carried out in future studies to establish the effects of herbs on Lactobacillus sp. and S. thermophilus, the key acid producing bacteria in the starter culture used. In the present study the initial pH value for M. piperita, O. basilicum and A. graveolenceyogurts did not differ much from plain-yogurt, indicating the acidic nature of the herbal extracts, if any, were minimal. The pH reduction rates for all herbal-yogurts between 90-180 minutes were higher than that for plain yogurt (Figure 4.1) and thus these herbal-yogurts took a shorter time to reach pH 4.5 compared to plain-yogurts. This points to the possibility that the presence of these herbs may have enhanced the proteolytic activity of Lactobacillus sp. and S. thermophilus. Continued refrigerated storage decreased the pH of yogurts to lower pH values (4.2-4.4) as a result of accumulation of acetic acid, acetaldehyde, formic acid and lactic acid. This which affected S. thermophilus which grows best at 5.5 may cease growing at pH 3.3-3.8 (Vedamuthu, 1978).

For all yogurt treatments, the rate of acid formation were higher than that for plain-yogurt during fermentation. Total titratable acidity is the total amount of hydrogen ions present in the fermented milk sample with the exception of those bound to alkaline ions. The determination of total titratable acid (TTA) thus is more relevant in the evaluation of fermentation capacity of microbes (Geidam et al., 2007).

5.2 Total phenolic content in herbal-yogurts.

Phenolic substances are a category of phytonutrients that exert strong antioxidant properties (Ho, et al 1992). They can be classified into simple phenols, phenolic acids, hydroxycinnamic acid derivatives and flavonoids. Among the variety of phenolic compounds, phenolic acids have attracted considerable. Due to their many potential health benefits. Phenolic acids are powerful antioxidants and have been reported to demonstrate antibacterial, antiviral, anticarcinogenic, anti-inflammatory and vasodilatory actions (Duthie et al., 2000; Breinholt, 1999; Shahidi and Naczk, 1995). The Folin- Ciocalteu method is a rapid and widely-used assay, to investigate the total phenolic content but it is known that different phenolic compounds have different responses in the Folin-Ciocalteu method (Kahkonen et al., 1999). Thus the comparison of total phenolic contents (TPC) between herbs, or herbal-yogurts may only be used qualitatively to a limited extent. Different TPC was observed in herbal-yogurts compared to plain-yogurt and it tends to increase for all yogurts during storage days. Since plain-yogurt contains no plant extracts, the TPC values reflect phenolic compounds from protein. Yogurt itself contains amino acid such as tyrosin that has side chain group the same as phenolic group which give rise to the reading in TPC (Shah, 2000). Another possibility is that microbial utilization of phenolic acids such as

ferulic and p-coumaric acid during fermentation process and post acidification lead to the production of other phenolic acids such as vanillic acid and p-hydroxybenzoic acid before the aromatic ring structure is broken (Turner and Rice, 1979; Blum, 1998). For herbal-yogurt however consideration must be given to the inherently high plant compounds. The high TPC A. graveolenve-yogurt for instance may be attributed to the presence of flavonoids and phenolic compounds (Ishikawa et al. 2002).

5.3 Antioxidant activities of herbal-yogurts.

The principle function of antioxidants is in delaying the oxidation of other molecules by inhibiting the initiation or propagation of oxidizing chain reactions by free radicals (Namiki, 1990). From food technologist point of view, an antioxidant is useful in delaying, retarding or preventing the development of rancidity or other off-flavour, due to oxidation at a low concentration, compared with that of the oxidizing substrate (Gordon., 1990). Since antioxidants can be classified according to their protective properties at different stages of the oxidation process and since they act by different mechanisms, they are divided into two main types of antioxidants: primary and secondary antioxidants. Primary antioxidants can inhibit or retard oxidation by scavenging free radicals by donation of hydrogen atoms or electrons, which converts them to more stable products. Secondary antioxidants function by many mechanisms, including binding of metal ions, scavenging oxygen, converting hydroperoxides to non-radical species, absorbing UV radiation or deactivating singlet oxygen (Gordon, 1990). The DPPH radical is a stable free radical and the DPPH radical-scavenging activity was determined by the decrease in absorbance at 517 nm, due to reduction by the antioxidant (AH) or reaction with a radical species (R) (Gordon, 1990), as decribed in the following equations.

DPPH + AH DPPH - H + A DPPH + R DPPH R

Plants contain a variety of naturally-occurring minor components (chemicals or substances) present in plants (Caragay, 1992) called phytochemicals (Pratt, 1992). These include flavonoids, tocopherols, carotenoids, and ascorbic acids (Meyers, et al, 2003). Extracts from plants which contribute health benefits to consumers, arising from protection from free radical-mediated deteriorations, and which cause retardation of lipid oxidation (Oktay,et al, 2003) had stronger antioxidant activity than that of synthetic antioxidants. Thus the making of herbal-yogurts may in its own right increased the antioxidant properties of yogurt. The present studies showed that each type of herbs had a different antioxidant activity, most likely contributed by each herbal phytochemical characteristics (see 2.5.1, 2.6.1 and 2.7.1). [For instance, A. graveolence contains flavanoids and tannins, whereas M. piperita and O. bacilicum contain high -tocopherol, -carotene and ferulic acid high in antioxidant activity (Ismail, et al., 2004).]. Highest antioxidant activity on day 7 recorded in A.graveolenceyogurt followed by M. piperita- , O. basilicum- and plain-yogurts may be attributed to the probiotics in yogurts. These microbes are still metabolically active even at low temperature. Continued microbial growth during refrigerated storage may have altered some of the phenolic compounds and hence their antioxidant activities (Blum and Shafer, 1988).

5.4 Proteolysis and changes in OPA peptides in herbal-yogurts

Peptides with biological activities, released during gastrointestinal digestion or food processing, can affect metabolic regulation and modulation. In fact bioactive peptides serve as

important nutraceuticals and functional food ingredients for health promotion and disease risk reduction. Many studies have reported that peptides from various food sources possess bioactivities, including antihypertensive, antioxidant, anticancer, antimicrobial, and opioid activities as well as immune modulatory and cholesterol-lowering effects (Shahidi & Zhong 2008). Fermentation of milk by LAB in different types of yogurts resulted in different amount of OPA peptides produced (see Figure 4.5). This reflects the extent of proteolysis as influenced by the presence of herbs. Even more interesting was the formation of more OPA peptides during the first 7 days of refrigerated storage. This could be due to the L. acidophilus and S. thermophilus, which are known to be metabolically active even at 4C. The higher amount of peptide in M. piperita-yogurt followed by A. graveolence- and O. basilicum-yogurts than that in plain-yogurt showed that the presence of these herbs increased the proteolytic activity of lactic acid bacteria in yogurt. The extent of proteolysis and the sizes of peptides produced from the fermentation are likely to be different for each treatment and this need to be further studied in the future. This is because peptides and amino acids are further degraded to yield carbon skeletons that are used as nutrients by LAB. This is demonstrated as the decline in the concentration of OPA peptides from day 7 to day 28 in all yogurts. It is important to note from present studies that the addition of certain herbs may regulate the extent of proteolysis which could have desirable effects with respect to the production of bioactive proteins (see Section 5.5).

5.5. Inhibition of angiotensin-1 converting enzyme (ACE) activity by herbalyogurts

The angiotensin I-converting enzyme (ACE) assay was carried out to evaluate the inhibitory activity of bioactive peptides produced during yogurt formation and refrigerated storage. Milk proteins, especially caseins, are an important source of these bioactive peptides. Milk protein contains ACE inhibitory peptides encrypted within their primary structures. These peptides can be released by enzymatic hydrolysis either during gastrointestinal digestion or during food processing. Peptides with ACE inhibitory activity have been isolated from different food protein (Yamamoto, 1997) and milk products fermented with LAB (Gobbetti et al, 2000). The ACE activity of plain-yogurt and herbal-yogurts do correlate well with OPA peptide whereby an increase in peptide concentration in yogurt increases the inhibition of ACE activity (See Table 4.1). All herbal yogurts showed anti-ACE activity higher than plain yogurt at respective storage periods. Also plain-yogurt and herbal-yogurts achieved their highest ACE inhibitor potentials on day 7 of storage (see figure 4.7) which again coincidentally indicate that higher ACE inhibitory activity related to higher proteolysis. It is possible that specific peptides generated during fermentation and refrigerated storage in the presence of herbs (M. piperita, A. graveolence, O. basilicum) has high anti-ACE activity and this is useful for hypertension management. Proteolysis is the most important biochemical process occurring during fermentation and storage (Tamine & Deeth, 1980). Fermented milk products which contain bioactive peptides valyl-prolyl-proline (Val-Pro-Pro) and isoleucyl-prolyl-proline (II-Pro-Pro) were shown to lower blood pressure in spontaneously hypertensive (Nakamura et al., 1995). These peptides act as ACE inhibitor which will bind to the enzyme competitively and prevent the breakdown of substrate, furanacrylyl-Phe-Gly-Gly (FAPGG) to the product, furanacryly-Phe (FAP) and GlyGly. This observation suggests that an increase in bioactive peptides in yogurt will decrease the

ACE activity i.e. an increase in the therapeutical value of yogurt compared to milk. The present studies showed that the addition of herbs (M. piperita, A. graveolence and O. basilicum) in yogurt increased the inhibition of ACE activity which can be correlated to increased degree of proteolysis during storage. Thus, future studies using HPLC and amino acid analysis are expected to yield interesting results with respect to the relationship of specific peptides produced in herbal-yogurts to ACE-inhibitory activities.

5.6 Inhibition of -amylase activity by herbal-yogurts

Many secondary chemical compounds synthesized by plants are associated with plant defence. These include enzyme inhibitors (Schuler et. al, 1998) which impede digestion through their action on insect gut digestive -amylases and proteinases, which play a key role in the digestion of plant starch and proteins (Ryan, 1990). The pancreatic -amylase (50000 mw) is the first enzyme which was found to be modulated by an effector ligand. The consumption of yogurt has been suggested to be beneficial to those with diabetes (Salminen, et al, 2005) and this is attributed partly to the anti-amylase activities. The present studies showed that A. graveolence and M. piperita-yogurts gave pronounced inhibition on -amylase activity whereas O. basilicumyogurt had less inhibitory effects (higher IC50) than that in plain-yogurt (see Figure 4.8) This finding implicate that the therapeutic benefit of yogurt with respect to anti-diabetes may be enhanced by adding certain herbs. It is of no surprise that many herbal medicines have been used in the prevention and treatment of diabetes and thus it is highly likely that the phytochemicals in these plants play important roles in regulating intestinal enzyme activities. The enhanced antiamylase activities of the herbal-yogurts, at least during the first 7 days of refrigerated storage,

points out the possibilities that the phytochemicals may be degraded to more active forms during storage. However, with regard to complete assessment of the benefits of herbal-yogurts

consumption as a mean to control diabetes, the effects of these yogurts on -glucosidase, another intestinal enzyme, important in diabetes need to be examined.

5.7 Inhibition of -glucosidase activity by herbal-yogurts

-glucosidase is an enzyme located at brush border of microvillus in small intestine. This enzyme is a regulatory enzyme where it catalyzes the final step of carbohydrate metabolism which is oligosaccharides to monosaccharides prior to absorption into the blood circulation (Kawabata et al., 2007). All herbal yogurts showed maximal inhibition on -glucosidase on day 7 of storage (see Figure 4.10) followed by gradual reduction inhibitory activity thereafter. Highest inhibition of -glucosidase on day 7 was shown by A. graveolence-yogurt followed by M. piperita- and O. basilicum-yogurt. The profound effects of these herbs may be attributed to flavonoids and polyphenols, as well as their sugar derivates which are known to be effective inhibitors of -glucosidase (Kaushik et al., 2008). Phenolic compounds are known inhibitors of -amylase and -glocosidase (McCue et al., 2005). A. graveolence has high inhibition on glucosidase and this may be explain by the high tannin (Yazdanparast and Alavi., 2001) which are polimerized form of phenols/ phenolics compound/polyphenols. In this regard, it would be interesting to demonstrate in future in vivo studies the effects of consumption of A. graveolenceyogurt on slowing the rise of blood sugar level after a meal.

5.8 General discussion

The present studies have demonstrated 2 important observations i.e. i) the presence of herbs has profound effect on the formation of yogurt by LAB and, ii) the refrigerated storage of yogurts resulted in changes in proteolysis and enzyme inhibitory capacities which to a large extent can be attributed by the presence of herbs. The popularity of yogurt as a functional food is very much related to its good taste, nutritional properties (rich in potassium, calcium, protein and vitamin B), better digestibility of proteins and potentially beneficial effects in human health by supplying pre-biotic and pro-biotic bacteria (Adolfsson, et al, 2004). The immediate health benefits yogurt includes to fortify the immune system, improves lactose digestion and gastrointestinal conditions (Kampman, et al, 1994) and allergies (Doleyres, et al, 2005) and lowering blood pressure of spontaneously hypertensive rats and humans (Nakamura, et al, 1995). These potentially diverse functional properties of yogurt have resulted in increased production of different types of milk and milk products to meet increasing variety of consumer demands and may include the mixing of fruits (Thompson, Lopetcharat and Drake, 2007), fiber (Fernndez-Gara, Mcgregor and Traylor, 1998; Sanz, Salvador, Jimnez & Fiszman (2008) and herbs (Rudkowska, et al, 2008; Ruggeri et al, 2008) rich in phytonutrients. In this regard, the present studies have provide further support of on the use of herbs to manipulate the formation of bioactive peptides in milk products with ACE, -amylase and -glucosidase inhibitory activities. The benefits of including herbs into yogurt may increased yogurt eating values in several ways. Firstly, the duration of fermentation may be reduced (6 hrs for herbal yogurts as opposed

to 7 hrs for plain yogurt; Figure 4.1). This could be an advantage to the industry with respect to operating hours and labour cost. Secondly, the addition of herb increased antioxidant levels of yogurts. Flavonoids are very effective antioxidants (Yanishlieva-Maslarova, 2001). These are large group of naturally-occurring plant phenolic compounds including flavones, flavonols, isoflavones, flavonones and chalcones. Flavonoids contain a characteristic C6-C3-C6 structure, with free hydroxyl groups attached to aromatic rings, and they inhibit lipid oxidation by scavenging radicals or by other mechanisms such as singlet oxygen quenching, metal chelation, and lipoxygenase inhibition (Yanishlieva- Maslarova, 2001). In addition, several flavonoids are also reported to act as antiulcer, antispasmodic, antisecretory, or antidiarrhoeal agents in the gastrointestinal tract (Carlo et al., 1999), and possess antihepatotoxic properties (Beretz and Cazenave, 1988; Eaton-Evans, 1994). These are some of the attributes commonly associated with the consumption of A. graveolance, O. basilicum and M. piperita (see Sections 2.5.2, 2.6.2 and 2.7.2). Thirdly, the potential therapeutically properties of yogurt, particularly the lowering of blood glucose and blood pressure, may be enhanced by the addition of selected herb. The higher peptide contents in herbal-yogurts may thus be attributed to the proteolytic activity from a combined high microbial counts of L. acidophilus and S. thermophillus (Baba and Najarian, in press). Big difference in IC50 values for ACE activity (plain-yogurt; Figures 4.7) and -amylase and -glucosidase (plain- and O. basilicum-yogurts; Figures 4.9 and 4.11) between fresh and day 7 refrigerated yogurts suggest that most of the peptides in fresh yogurts were in less bioactive forms. Hence the increased formation of bioactive peptides (reduction in IC50 values) during the first 7 days of refrigerated storage may be attributed to the proteolysis of relatively nonspecific peptides produced during fermentation into more bioactive peptides during this period. Such an

observation is not new as it is widely known that major milk protein groups, caseins and whey proteins, are an important source of peptides with biological activities e.g., antihypertensive, antimicrobial, immunomodulatory, opioid or mineral binding activities. These peptides are in an inactive state inside a protein molecule and can be released during enzymatic hydrolysis with digestive enzymes ( Hernandez-Ledesma, Amigo, Ramos and Recio (2004), fermentation of milk by proteolytic starter cultures (Korhonen & Pihlanto- Leppa, 2001), or through the action of enzymes derived from proteolytic microorganisms (Maeno et al, 1996). Since the products of proteolysis also increased during refrigerated storage, and that OPA peptides correlates well with anti-ACE activity, one may also consider that these peptides may also cause the increase in -amylase and -glucosidase inhibition. Such possibility however may be excluded, at least by the observation that there were weak correlations (Table 4.1) between the level of OPA peptides and -amylase or -glucosidase. It is a common clinical practice to prescribe -glucosidase inhibitors such as acarbose and miglitol. Both drugs interfere with the action of the -glucosidase present in the small intestinal brush border (Kawabata et al., 2007). However the inhibition may be too strong that the carbohydrate may be passed on to be fermented by microflora in the large intestine. Taken as a whole, the consumption of herbal-yogurts, particularly A. graveolance- and M. piperita-yogurts may slow down the digestion of ingested carbohydrates by inhibiting both -amylase and glucosidase. This is in contrast to other reports (Kwon et al., 2006) which showed natural amylase and -glucosidase inhibitors from plant had lower inhibitory effect against -amylase activity but a stronger inhibition activity against -glucosidase. The difference can be attributed to the phytochemicals present in these plants that affect one enzyme more than the other. In either case, both findings strongly support the potential uses of natural compounds as effective

therapy for postprandial hyperglycemia with minimal side effects. This may prove useful for improving glycemic control in subjects suffering from borderline and type-2 diabetes mellitus. Shelf life of refrigerated commercial yogurts is usually regarded to be as long as 14-21 days post-fermentation. The present studies highlights strong evidence that the shelf life of yogurts depend very much on the functional values that one requires of yogurt. For ACEinhibitory activities yogurt should be consumed at about 72 days of refrigerated storage (Figure 4.6) whereas for -amylase-inhibition activities yogurts should be consumed between 7 and 14 days of storage (Figure 4.8). -glucosidase inhibitory levels in yogurts appeared to be still high even on day 21 of storage (Figure 4.10), and as such may be consumed even as late as until 3 weeks post-production. However, since the consumption of yogurt is normally for the benefits of probiotics contained in it, and since probiotics have very limited refrigerated shelf life (optimally 14 days), a reasonable compromise of between 7 to 18 refrigerated storage days is recommended in order to obtain the optimum benefits of yogurt eating. The aforementioned advantages of herbal-yogurts merit more research in the future. Important aspects of commercial yogurt production such as organoleptic and microbiological properties need to be ascertained prior to considering pilot run on the potential consumer acceptability. For instance, considerable negative impact of the enhanced proteolysis on the organoleptic property of yogurt (Matoba et al, 1970) as a result of the addition of herbs is anticipated and this may be a minus point. Nevertheless, evaluation of organoleptic properties is very subjective since undesireable tastes to some may be desirable to others.

5.9 Conclusion

Addition of herbs into yogurts altered the fermentation of milk. The presence of A. graveolence into yogurt increased inhibitory effects on in vitro -amylase and -glucosidase, total phenolic content, and antioxidant activity. M. piperita increased proteolysis activity which directly enhanced the inhibitory effect on ACE activity. A. graveolence-, M. piperita- and O. basilicum-yogurts may have practical application in the regulators of intestinal absorption of glucose and blood pressure.

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Appendices
Appendix 1: pH analysis pH value of yogurts during fermentation First Batch time 0 30 60 90 120 150 180 210 240 270 300 330 360 390 420 plain 6.52 6.31 6.22 6.18 6.11 5.87 5.5 5.23 4.97 4.9 4.83 4.72 4.65 4.38 4.37 ocimum 6.49 6.29 6.23 6.35 5.41 5.2 4.96 4.81 4.69 4.6 4.46 4.32 4.29 4.28 peppermint 6.51 6.42 6.31 5.92 5.41 5.23 4.92 4.81 4.68 4.57 4.48 4.39 4.35 4.3 gravolence 6.45 6.38 6.25 5.85 5.26 5.01 4.81 4.67 4.54 4.44 4.37 4.32 4.28 4.25

Second Batch time 0 30 60 90 120 150 180 210 240 270 300 330 360 390 420 plain 6.49 6.37 6.27 6.19 6.1 5.83 5.61 5.35 5 4.92 4.8 4.71 4.62 4.49 4.45 ocimum 6.46 6.32 6.21 6.04 5.83 5.57 5.12 4.96 4.74 4.63 4.51 4.44 4.3 4.28 peppermint 6.48 6.41 6.3 6.07 5.74 5.52 5.11 4.92 4.79 4.68 4.5 4.43 4.34 4.32 gravolence 6.44 6.33 6.2 5.85 5.54 5.03 4.82 4.69 4.51 4.44 4.33 4.27 4.25 4.21

Third Batch time 0 30 60 90 120 150 180 210 240 270 300 330 360 390 420 plain 6.5 6.4 6.33 6.25 6.15 5.9 5.78 5.56 5.2 4.93 4.8 4.67 4.52 4.39 4.38 ocimum 6.4 6.28 6.17 5.84 5.7 5.54 5.15 4.96 4.81 4.69 4.52 4.39 4.29 4.27 peppermint 6.43 6.38 6.25 6.09 5.99 5.64 5.3 5.014 4.85 4.67 4.53 4.42 4.3 4.36 gravolence 6.38 6.23 6.15 5.82 5.64 5.37 4.93 4.72 4.5 4.43 4.37 4.29 4.25 4.23

Appendix 2: Total Titrable Acidity (TTA) analysis Volume of NaOH(ml)used in TTA analysis

First Batch time 0 30 60 90 120 150 180 210 240 270 300 330 360 390 420 plain 0.2 0.25 0.3 0.35 0.4 0.45 0.5 0.5 0.6 0.65 0.7 0.75 0.8 0.85 0.85 ocimum 0.2 0.25 0.3 0.35 0.4 0.45 0.5 0.6 0.7 0.75 0.8 0.85 0.9 0.9 peppermint 0.2 0.25 0.3 0.35 0.4 0.45 0.5 0.55 0.6 0.65 0.7 0.75 0.8 0.8 gravolence 0.2 0.25 0.3 0.4 0.5 0.55 0.6 0.7 0.8 0.85 0.9 0.95 0.95 0.9

Second Batch time 0 30 60 90 120 150 180 210 240 270 300 330 360 390 420 plain 0.2 0.2 0.2 0.3 0.35 0.4 0.45 0.5 0.55 0.6 0.65 0.7 0.75 0.8 0.85 ocimum 0.2 0.2 0.25 0.3 0.35 0.4 0.45 0.5 0.55 0.6 0.65 0.7 0.75 0.8 peppermint 0.2 0.25 0.3 0.35 0.4 0.45 0.5 0.55 0.6 0.65 0.7 0.75 0.8 0.85 gravolence 0.2 0.25 0.3 0.35 0.4 0.45 0.5 0.6 0.7 0.75 0.8 0.85 0.9 0.95

Third Batch time 0 30 60 90 120 150 180 210 240 270 300 330 360 390 420 plain 0.2 0.2 0.25 0.3 0.35 0.4 0.4 0.45 0.5 0.55 0.6 0.65 0.7 0.75 0.8 ocimum 0.3 0.3 0.35 0.4 0.45 0.5 0.55 0.55 0.6 0.65 0.7 0.8 0.85 0.85 peppermint 0.2 0.25 0.3 0.35 0.4 0.45 0.5 0.55 0.6 0.65 0.7 0.75 0.8 0.85 gravolence 0.3 0.35 0.4 0.45 0.5 0.55 0.6 0.7 0.75 0.8 0.85 0.9 0.95 0.95

Appendix 3: DPPH assay Absorbance reading(517nm) of DPPH assay at different storage days First Batch 0 control plain ocimum peppermint gravolence Second Batch 0 control plain ocimum peppermint gravolence Third Batch 0 control plain ocimum peppermint gravolence 0.459 0.322 0.304 0.229 0.207 7 0.466 0.316 0.256 0.219 0.212 14 0.459 0.328 0.268 0.24 0.19 21 0.471 0.341 0.3 0.22 0.2 28 0.47 0.35 0.305 0.245 0.268 0.473 0.328 0.31 0.229 0.197 7 0.464 0.309 0.232 0.216 0.175 14 0.45 0.314 0.258 0.22 0.183 21 0.466 0.337 0.308 0.29 0.249 28 0.47 0.341 0.311 0.231 0.253 0.456 0.31 0.29 0.235 0.19 7 0.463 0.29 0.255 0.214 0.177 14 0.47 0.315 0.264 0.238 0.179 21 0.445 0.32 0.302 0.29 0.24 28 0.466 0.329 0.305 0.298 0.246

Percentage inhibition (%) of DPPH at different storage days

First Batch 0 plain ocimum peppermint gravolence 32.02 36.40 48.46 58.33 7 33.76 41.32 50.17 58.16 14 32.98 43.83 49.36 61.91 21 28.09 32.13 34.83 46.07 28 29.40 34.55 36.05 47.21

Second Batch 0 plain ocimum peppermint gravolence 30.66 34.46 51.59 58.35 7 33.41 50.00 53.45 62.28 14 30.22 42.67 51.11 59.33 21 27.68 33.91 37.77 46.57 28 27.45 33.83 50.85 46.17

Third Batch 0 plain ocimum peppermint gravolence 29.85 33.77 50.11 54.90 7 32.19 45.06 53.00 54.51 14 28.54 41.61 47.71 58.61 21 27.60 36.31 53.29 57.54 28 25.53 35.11 47.87 42.98

Appendix 4: Total Phenolic assay Absorbance reading(725nm) for total phenolic assay

First Batch 0 plain ocimum peppermint gravolence 0.312 0.38 0.46 0.69 7 0.327 0.412 0.53 0.75 14 0.331 0.48 0.53 0.83 21 0.345 0.53 0.61 0.89 28 0.387 0.58 0.67 0.92

Second Batch 0 plain ocimum peppermint gravolence 0.29 0.39 0.5 0.65 7 0.31 0.45 0.65 0.79 14 0.323 0.44 0.66 0.87 21 0.344 0.48 0.71 0.79 28 0.355 0.51 0.75 0.89

Third Batch 0 plain ocimum peppermint gravolence 0.32 0.43 0.6 0.74 7 0.339 0.56 0.71 0.79 14 0.348 0.47 0.77 0.89 21 0.37 0.55 0.78 0.91 28 0.412 0.58 0.76 0.95

total phenolic content(g/g) of herbal yogurts during storage

First Batch 0 plain ocimum peppermint gravolence 15.68 19.26 23.47 35.58 7 16.47 20.95 27.16 38.74 14 16.68 24.53 27.16 42.95 21 17.42 27.16 31.37 46.11 28 19.63 29.79 34.53 47.68

Second Batch 0 plain ocimum peppermint gravolence 14.53 19.79 25.58 33.47 7 15.58 22.95 33.47 40.84 14 16.26 22.42 34.00 45.05 21 17.37 24.53 36.63 40.84 28 17.95 26.11 38.74 46.11

Third Batch 0 plain ocimum peppermint gravolence 16.11 21.89 30.84 38.21 7 17.11 28.74 36.63 40.84 14 17.58 24.00 39.79 46.11 21 18.74 28.21 40.32 47.16 28 20.95 29.79 39.26 49.26

Gallic acid standard curve Concentartion g/ml 5 10 20 30 40 50 60 Abs reading 0.123 0.23 0.39 0.601 0.764 1.032 1.204

Appendix 5: OPA assay

absorbance reading(340nm) of yogurt at different storage days

First Batch 0 plain ocimum peppermint gravolence 0.357 0.441 0.46 0.48 7 0.411 0.723 0.749 0.688 14 0.393 0.655 0.729 0.635 21 0.389 0.612 0.7 0.512 28 0.32 0.508 0.587 0.533

Second Batch 0 plain ocimum peppermint gravolence 0.34 0.51 0.48 0.459 7 0.404 0.695 0.75 0.75 14 0.399 0.69 0.689 0.622 21 0.402 0.51 0.59 0.603 28 0.333 0.467 0.65 0.549

Third Batch 0 plain ocimum peppermint gravolence 0.408 0.488 0.53 0.498 7 0.456 0.644 0.77 0.705 14 0.42 0.599 0.677 0.646 21 0.4 0.5 0.583 0.615 28 0.312 0.433 0.6 0.541

Peptide concentration (mg/g) of yogurts at different storage days

First Batch 0 plain ocimum peppermint gravolence 14.958 18.840 19.718 20.642 7 17.454 31.872 33.073 30.254 14 16.622 28.729 32.149 27.805 21 16.437 26.742 30.809 22.121 28 13.249 21.936 25.587 23.091

Second Batch 0 plain ocimum peppermint gravolence 14.173 22.029 20.642 19.672 7 17.130 30.578 33.119 33.119 14 16.899 30.347 30.300 27.204 21 17.038 22.029 25.726 26.326 28 13.849 20.042 28.498 23.831

Third Batch 0 plain ocimum peppermint gravolence 17.315 21.012 22.953 21.474 7 19.533 28.221 34.043 31.040 14 17.870 26.141 29.746 28.313 21 16.945 21.567 25.402 26.881 28 12.879 18.470 26.188 23.461

Tryptone standard curve

Concentration mg/g 0.25 0.5 0.75 1 1.25 1.5

Abs reading 0.185 0.306 0.398 0.588 0.724 0.844

Appendix 6: ACE inhibition assay

percentage of inhibition(%)of ACE at different storage day(300l) First Batch 0 control plain ocimum peppermint gravolence Second Batch 0 control plain ocimum peppermint gravolence Third Batch 0 control plain ocimum peppermint gravolence 0.0136 45.89 61 83 69 7 0.0134 60.49 66 90 78 14 0.0129 59.12 62 87 72.26 21 0.013 46.03 52 70 63.88 28 0.0129 19.55 43 54 43.1 0.0136 42.09 56 87 75 7 0.0134 67.54 64.84 93 66.4 14 0.0129 51 61 92 59.63 21 0.013 42 52 77 50.41 28 0.0129 29.88 47 69 44.87 0.0136 45 63 82 56 7 0.0134 63 70 90 78 14 0.0129 51 65.4 83 65 21 0.013 40 59.2 75 56.9 28 0.0129 37 53.8 67.9 52.3

percentage of inhibition(%)of ACE at different storage day(150l) First Batch 0 control plain ocimum peppermint gravolence Second Batch 0 control plain ocimum peppermint gravolence Third Batch 0 control plain ocimum peppermint gravolence 0.0136 20.59 25.45 55.09 35.6 7 0.0134 36.09 37.71 50.67 40.08 14 0.0129 24.15 33 58.8 42 21 0.013 19.64 26 42 25 28 0.0129 7.89 16.09 31.99 17 0.0136 26.85 27.34 47.12 37.98 7 0.0134 33.32 36.45 58.72 43 14 0.0129 29.09 31.99 53 42.51 21 0.013 19.43 25.8 46 26.05 28 0.0129 14.67 19.44 32.11 16.6 0.0136 23 25.7 63.66 31 7 0.0134 29 30.11 69.15 37 14 0.0129 23 23.8 61.02 34 21 0.013 21 20 47.69 17.3 28 0.0129 19 19.4 39.7 16

percentage of inhibition(%)of ACE at different storage day(75l)

First Batch 0 control plain ocimum peppermint gravolence 0.0136 13.12 15.77 26 18 7 0.0134 18.77 19 32 18 14 0.0129 15.91 12.6 28 14 21 0.013 9.74 11 14 12 28 0.0129 7.39 8.6 10 9

Second Batch 0 control plain ocimum peppermint gravolence 0.0136 12.78 14 23.08 25.5 7 0.0134 22.89 20.94 35 33 14 0.0129 15.99 18.05 37.11 28.44 21 0.013 8.99 12.34 20.06 13 28 0.0129 6.56 8.99 14 10

Third Batch 0 Control Plain Ocimum Peppermint Gravolence 0.0136 11.37 12.05 25.09 22.9 7 0.0134 15.26 19.91 25.06 31 14 0.0129 16.03 12 23.88 25 21 0.013 7.09 10.02 21 18 28 0.0129 2.98 8.5 15 8.024

Absorbance reading (340)- Enalaprill

0.00 5.00 10.00 15.00 20.00 Abs5'-20'

-4.00 1.48 1.48 1.48 1.47 1.47

-5.00 1.44 1.44 1.44 1.44 1.44

-6.00 1.43 1.42 1.42 1.42 1.41 0.01

-7.00 1.43 1.41 1.40 1.39 1.38 0.03

-8.00 1.42 1.38 1.35 1.33 1.30 0.83

-9.00 1.39 1.35 1.31 1.28 1.24 0.12

-10.00 1.33 1.28 1.21 1.16 1.13 0.15

-11.00 1.32 1.26 1.19 1.12 1.08 0.17

-12.00 1.23 1.20 1.14 1.08 1.01 0.19

Determination of optimal rabbit lung (blank) dilution factor time 0 5 10 15 20 Abs5'-20' standard 2.01 1.88 1.80 1.73 1.64 0.24 2x 1.85 1.68 1.56 1.50 1.48 0.20 4x 1.48 1.32 1.23 1.16 1.12 0.19 5x 1.40 1.27 1.17 1.12 1.08 0.19 7x 1.33 1.22 1.16 1.08 1.03 0.18 10x 1.23 1.16 1.13 1.07 1.00 0.16

Appendix 7: Alpha amylase inhibition assay

First batch 500 control plain peppermint ocimum gravolence 0 0.368 0.317 0.253 0.305 0.196 7 0.37 0.264 0.215 0.237 0.157 14 0.374 0.25 0.231 0.264 0.166 21 0.345 0.322 0.247 0.281 0.188 28 0.336 0.324 0.266 0.322 0.2

Second batch 500 control plain peppermint ocimum gravolence 0 0.357 0.267 0.245 0.305 0.183 7 0.337 0.305 0.209 0.211 0.166 14 0.343 0.291 0.221 0.256 0.172 21 0.355 0.2883 0.249 0.296 0.203 28 0.347 0.269 0.259 0.319 0.218

Third batch 500 control plain peppermint ocimum gravolence 0 0.35 0.31 0.244 0.268 0.189 7 0.351 0.247 0.214 0.22 0.123 14 0.343 0.29 0.22 0.23 0.167 21 0.362 0.298 0.256 0.306 0.188 28 0.342 0.324 0.267 0.256 0.21

First batch

250 Control Plain peppermint ocimum gravolence

0 0.362 0.312 0.278 0.317 0.256

7 0.311 0.273 0.256 0.292 0.243

14 0.315 0.297 0.261 0.311 0.269

21 0.328 0.322 0.285 0.292 0.287

28 0.327 0.309 0.213 0.286 0.291

Second batch 250 Control Plain Peppermint Ocimum Gravolence 0 0.337 0.309 0.291 0.275 0.244 7 0.341 0.265 0.284 0.281 0.245 14 0.351 0.289 0.245 0.288 0.256 21 0.33 0.307 0.262 0.328 0.277 28 0.299 0.309 0.271 0.291 0.262

Third batch 250 control plain peppermint ocimum gravolence 0 0.3 0.289 0.289 0.298 0.267 7 0.326 0.34 0.253 0.243 0.21 14 0.344 0.301 0.252 0.267 0.28 21 0.341 0.29 0.269 0.306 0.233 28 0.34 0.3 0.277 0.336 0.255

First batch

125 control plain peppermint ocimum gravolence

0 0.318 0.285 0.304 0.281 0.273

7 0.333 0.297 0.322 0.271 0.266

14 0.299 0.282 0.288 0.283 0.271

21 0.333 0.293 0.3018 0.281 0.255

28 0.336 0.274 0.298 0.284 0.283

Second batch 125 control plain peppermint ocimum gravolence 0 0.34 0.279 0.245 0.317 0.261 7 0.288 0.28 0.241 0.328 0.274 14 0.305 0.28 0.21 0.287 0.263 21 0.325 0.28 0.27 0.304 0.267 28 0.312 0.258 0.237 0.325 0.272

Third batch 125 control plain peppermint ocimum gravolence 0 0.34 0.307 0.288 0.257 0.269 7 0.32 0.312 0.28 0.253 0.273 14 0.286 0.282 0.288 0.251 0.278 21 0.305 0.288 0.234 0.279 0.284 28 0.31 0.278 0.285 0.286 0.289

Appendix 8: Alpha glucosidase inhibition assay

batche 1 500 l 0
before after

7
before after

14
before after

21
before after

28
before after

control plain ocimum peppermint gravolence

0.06 2.71 0.05 2.7 0.06 2.47 0.06 2.27 0.05 2.2

0.06 0.03 0.08 0.09 0.09

2.83 2.47 2.63 2.18 1.82

0.09 0.04 0.07 0.06 0.07

2.97 2.83 2.44 2.29 1.89

0.06 0.07 0.05 0.05 0.05

2.71 2.59 2.49 2.11 2.39

0.1 0.08 0.07 0.06 0.06

2.53 2.48 2.42 2.29 2.1

Batch 2 500l 0
before after

7
before after

14
before after

21
before after

28
before after

control plain ocimum peppermint gravolence

0.08 0.05 0.06 0.07 0.06

2.93 2.63 2.55 2.49 2.51

0.03 0.07 0.08 0.08 0.08

2.74 2.44 2.29 2.29 1.85

0.06 0.05 0.07 0.06 0.07

2.83 2.67 2.41 2.31 2

0.04 2.84 0.03 2.7 0.05 2.4 0.05 2.06 0.06 2.24

0.07 2.88 0.05 2.8 0.06 2.6 0.05 2.42 0.06 2

Batch 3 500l 0
before after

7
before after

14
before after

21
before after

28
before after

control plain ocimum peppermint gravolence

0.09 0.05 0.05 0.06 0.07

3.06 2.68 2.73 2.69 2.45

0.05 0.04 0.06 0.05 0.07

3.15 2.56 2.52 2.38 1.81

0.04 0.06 0.07 0.05 0.08

2.95 2.4 2.47 2.33 2.17

0.05 0.06 0.05 0.07 0.06

2.76 2.61 2.32 2.1 2.09

0.05 0.06 0.06 0.07 0.07

2.68 2.43 2.31 2.53 2

batche 1

250 0
before after

7
before after

14
before after

21
before after

28
before after

control plain ocimum peppermint gravolence

0.1 2.63 0.05 2.6 0.06 2.38 0.06 2.4 0.07 2.34

0.04 0.06 0.06 0.07 0.06

2.97 2.7 2.84 2.68 2.55

0.04 0.05 0.05 0.06 0.06

3.61 3.53 3.21 3.16 2.99

0.06 0.07 0.06 0.06 0.08

2.81 2.72 2.62 2.4 2.53

0.06 2.95 0.07 2.88 0.05 2.8 0.06 2.79 0.06 2.2

Batch 2 250 0
before after

7
before after

14
before after

21
before after

28
before after

control plain ocimum peppermint gravolence

0.07 0.04 0.06 0.06 0.05

2.81 2.59 2.59 2.56 2.45

0.07 0.02 0.07 0.06 0.06

2.74 2.39 2.42 2.42 2.21

0.06 0.07 0.09 0.07 0.06

2.27 2.05 2.06 2 2.02

0.05 0.01 0.06 0.07 0.05

2.82 2.67 2.59 2.29 2.56

0.06 0.05 0.06 0.07 0.07

2.9 2.88 2.63 2.66 2.38

Batch 3 250 0
before after

7
before after

14
before after

21
before after

28
before after

control plain ocimum peppermint gravolence

0.05 0.06 0.07 0.06 0.07

3.19 2.86 2.87 2.99 2.83

0.09 0.05 0.08 0.06 0.06

3 2.54 2.56 2.59 2.58

0.06 2.38 0.09 2.1 0.07 2.1 0.09 2.05 0.09 2.1

0.05 0.06 0.05 0.06 0.07

2.78 2.7 2.39 2.18 2.52

0.06 0.03 0.06 0.05 0.06

2.79 2.57 2.7 2.58 2.33

first 125 0
before after

7
before after

14
before after

21
before after

28
before after

control plain ocimum peppermint gravolence

0.07 0.06 0.07 0.05 0.06

2.73 2.64 2.56 2.62 2.8

0.05 0.06 0.06 0.08 0.07

2.97 2.8 2.82 2.74 2.59

0.06 0.07 0.08 0.06 0.05

3.01 3 2.66 2.53 2.54

0.04 0.04 0.05 0.05 0.06

2.72 2.69 2.35 2.18 2.59

0.1 0.09 0.07 0.06 0.05

2.87 2.83 2.48 2.78 2.46

second 125 0
before after

7
before after

14
before after

21
before after

28
before after

control plain ocimum peppermint gravolence

0.06 0.04 0.05 0.06 0.06

2.88 2.73 2.78 2.74 2.52

0.07 0.04 0.05 0.06 0.06

2.77 2.41 2.43 2.36 2.65

0.08 0.06 0.05 0.06 0.06

2.31 2.18 2.11 2.08 2.28

0.04 0.06 0.06 0.07 0.05

2.96 2.9 2.86 2.59 2.76

0.05 2.9 0.07 2.83 0.06 2.9 0.05 2.87 0.07 2.11

third 125 0
before after

7
before after

14
before after

21
before after

28
before after

control plain ocimum peppermint gravolence

0.05 3.2 0.05 3 0.06 2.92 0.07 3 0.07 2.87

0.06 0.06 0.05 0.06 0.06

2.88 2.59 2.64 2.62 2.67

0.07 2.73 0.05 2.5 0.05 2.43 0.06 2.5 0.06 2.5

0.05 0.05 0.06 0.07 0.07

2.68 2.61 2.64 2.27 2.52

0.05 2.9 0.06 2.89 0.06 2.9 0.07 2.8 0.07 2.54

Appendix 9: Before yogurt fermentation (milk) Total phenolic content (g/ml)

batch1 plain M. piperita A. graveolence O. basilicus 12.21 18.53 28.00 11.18

batch2 13.80 16.28 26.30 13.78

batch3 12.15 19.01 33.50 15.78

mean 12.72 17.94 29.27 13.58

std 0.94 1.46 3.76 2.31

DPPH Inhibition Assay (% inhibition) batch1 plain M. piperita A. graveolence O. basilicus 28.14 39.40 50.89 32.04 batch2 26.12 41.53 55.63 29.32 batch3 28.78 41.77 53.46 33.76 mean 27.68 40.90 53.33 31.71 std 1.39 1.30 2.37 2.24

OPA Assay data (Peptide content, mg/ml)

batch1 plain M. piperita A. graveolence O. basilicus 10.21 20.55 22.73 16.63

batch2 9.88 18.43 21.22 16.95

batch3 11.45 16.77 19.07 17.65

mean 10.51 18.58 21.01 17.08

std 0.83 1.89 1.84 0.52

ACE Inhibition Assay

batch1 plain M. piperita A. graveolence O. basilicus 393.20 167.13 232.71 291.38

batch2 401.02 171.33 212.80 282.04

batch3 392.11 168.31 225.06 284.20

mean 395.44 168.92 223.52 285.87

std 4.86 2.17 10.04 4.89

alpha amylase inhibition assay (IC50 ,mg/g) batch1 plain peppermint graveolence ocimum 119.10 84.37 75.12 98.87 batch2 115.03 80.09 74.01 102.51 batch3 126.66 87.53 81.18 100.17 mean 120.26 84.00 76.77 100.52 std 5.90 3.73 3.86 1.84

alpha glocusidase inhibition assay (IC50 mg/g) batch1 plain peppermint graveolence ocimum 386.58 127.08 122.38 290.08 batch2 390.86 135.62 118.04 286.08 batch3 399.79 139.78 125.09 281.34 mean 392.41 134.16 121.84 285.83 std 6.74 6.47 3.56 4.38