INTRODUCTION

Lactic acid bacteria usually found in decomposition of plants and lactic products, produce lactic acid as the major metabolic end-product of carbohydrate fer- mentation. Lactobacillus species are probably the most important bacteria in the food industry. They are widely used as starter cultures and have been reported to play significant roles in the production of fermented foods. In addition to their importance in food fermentation, Lactobacillus species are generally recognized as safe

ARTICLE INFORMATION:

*Corresponding Author Received 18th April, 2015

Accepted after revision 15th May, 2015 BBRC Print ISSN: 0974-6455

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and have been reported to have beneficial health proper- ties, as a result of which they are also finding increasing use as probiotics in other health-related applications.

Whey, also called milk serum or milk permeate, is the liquid remaining after milk has been curdled and strained. It is a by-product of the manufacture of cheese or casein and has several commercial uses. Liquid whey contains lactose, vitamins, protein, and minerals, along with traces of fat. Whey appears to stimulate insulin release, in type 2 diabetes, (Lee et al., 1994, Jain et al., 2009 Fujiwara et al., 2004, Apostolids et al., 2006).

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Meena Vankudre, Anurup Balpande and Muktesh Athale

Oxidative damage plays a significantly pathological role in human diseases. Cancer, emphysema, cirrhosis and arthritis have all been correlated with oxidative damage. Highly reactive free radicals especially oxygen derived radicals which are formed by exogenous chemi- cals or endogenous metabolic processes in the human body or in food systems are capable of oxidizing bio- molecules resulting in cell death and tissue damage, (Auborg 1993, Baker and He,1991, Dave and Shah, 1996, Ramachandran and Shah 2006, and Tadere et al., 2006).

Synthetic antioxidants exhibit strong antioxidant activity against several oxidation systems. However, syn- thetic antioxidants pose potential risks in vivo; their use in food is restricted or prohibited in some countries. Anti- oxidants from natural sources are likely to be more desir- able than those chemically produced because some syn- thetic antioxidants have been reported to be side effects, (

Amylase inhibitors are also known as starch block- ers because they contain substances that prevent dietary starch from being absorbed by the body. Starch is com- plex carbohydrates that cannot be absorbed unless they are first broken down by the amylase enzyme. The inhi- bition of α-amylase enzyme delays carbohydrate diges- tion and protract overall carbohydrate digestion time, resulting in the reduction in glucose absorption rate and consequently dulling the postprandial plasma glucose rise. This study was carried out to evaluate inhibitory effect of Whey on pancreatic amylase activity.

MATERIAL AND METHODS

ISOLATION OF BACTERIA

Pure culture of Lactobacillus lactis and Lactobacillus delbeurkii was obtained from National Chemical Labora- tory, Pune and maintained on MRS agar plates in desic- cators for microaerophilic conditions.

INOCULATION OF BACTERIA INTO MILK

The bacterial suspension was prepared aseptically in suspension tube and the O.D was calculated using colo- rimeter. 2% of starter culture added into each flask con- taining skimmed milk. The milk was then incubated at room temperature.

PREPARATION OF WHEY FRACTION FROM FERMENTED MILK

Whey fraction was prepared as described by Virtanen et al., (2007). Whey separated from casein (15 ml) was collected from fermented milk and the pH was adjusted to 4.6 by adding 1M HCl. The suspension was centri- fuged (10,000g for 20 min at 5OC), and the supernatant was filtered on a micro filter.

DPPH RADICAL SCAVENGING ACTIVITY

Radical scavenging activity was estimated by method of Osuntoki et al., (2010). A volume of 2 mL of DPPH in

ethanol was added to 2 mL of the whey fraction, mixed and incubated in dark at room temperature for 30 min. The absorbance was measured at 517 nm by using UV spectrophotometer. Ethanol was used as a blank, while DPPH solution in ethanol served as the control. The rad- ical scavenging activity of the samples was expressed as

%inhibition of DPPH absorbance: Inhibition= [(Acontrol–Atest)/Acontrol] x100

Where Acontrol is the absorbance of the control sample (DPPH solution without whey fraction) and Atest is the absorbance of test sample (DPPH solution plus whey fraction).

α-AMYLASE INHIBITORY TEST:

Inhibition of α-amylase was evaluated by using method of Shori et al., (2014).Whey fraction (500μl) was added into 500 μl of phosphate buffer(0.02M) pH 6.9,contain- ing 0.5 mg/ml α-amylase solution were pre incubated at 25OC for 10 min. Starch solution (1% w/v in 0.02M sodium phosphate buffer, pH 6.9 with 0.006 M sodium chloride, 500 μl) was then added followed by incubation at 25OC for 10 min. The reaction was stopped by mixing with 1.0 ml of dinitrosalicylic acid (DNSA) color reagent. The test tubes were then incubated in a boiling water bath for 7 min. The absorbance reading was measured at 540 nm and compared to control which consists of 500 μl of buffer solution in place of the whey fraction. The enzyme inhibition was calculated as follows:

Inhibition (%) = [(Acontrol _ A extracts) / A control] X 100

RESULTS

DPPH RADICAL SCAVENGING ACTIVITY:

Buffalo milk whey fermented with L.lactis showed 32.73% inhibition which is higher as compared to cow milk whey, whereas cow milk whey fermented with L. delbeurkii showed 29.01% inhibition, greater than buf- falo milk whey.

α-AMYLASE INHIBITORY TEST:

The inhibitory activity of cow and buffalo milk whey fermented with LAB against α-amylase was studied. The comparison of inhibitory activity showed that L. del- beurkii has highest inhibition in both cow and buffalo milk whey.

Meena Vankudre, Anurup Balpande and Muktesh Athale

Similar results have been demonstrated by earlier workers like that of Duda-chodak and Tarko (2008) and Lara-Villoslada et al.,(2005)

Natural defense mechanisms eliminate negative effects of the activity of free radicals. However, they are not always adequate to totally neutralize all endogenous and exogenous free radicals. Thus, scavenging proper- ties of free radicals by food grade cultures can be use- ful in food manufacturing and can provide additional dietary sources of health enhancing antioxidants, (Shori 2013, Shori and Baba 2014).

In addition, a recent study has reported the develop- ment of antioxidant activity in whey during milk fermen- tation with lactic acid bacteria (Osuntoki et al., 2010).

The results indicate that the fermented milk whey show good fraction of antioxidant and α-amylase inhi- bition activity. The highest antioxidant activity was observed in the whey from buffalo milk fermented with the strain of L. lactis. This might be due to the strain being better adapted to the milk substrate, as compared to the other used strains.

Several studies have reported antioxidant activities in lactobacilli (Jain et al., 2009) a direct comparison of results is difficult because of the variety of assay meth- ods used, the numerous ways in which the results are expressed, the use of no standardized inoculums size. To increase glucose absorption in body α-amylase activity is important (Shori et al., 2011).

CONCLUSION

A reproducible protocol for production of milk whey using L. lactis and L. delbeurkii was standardizing.

In DPPH radical scavenging activity L. delbeurkii has activity in cow milk whey and L. lactis has higher activ- ity in buffalo milk whey, whereas in α-amylase inhibi- tion in both cow and buffalo milk L. deulburkii shows higher inhibition.

The whey of fermented skim milk has the potential to be a good dietary supplement into food formulations or cosmetics for prevention of oxidative stress related diseases such as atherosclerosis, coronary heart disease and cancer, and also useful to treat diabetes.

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