Almost all free and packaged foods and drinks undergo gradual changes during storage. Ignoring the degradation due to microorganisms, the typical cause of spoiling is the presence of oxygen and the products of chemical oxidation. The process of auto oxidation and the development of rancidity involve a free radical chain mechanism with several steps. In addition, lipid quality deteriorates under photo oxidative conditions or oxidation under thermal conditions such as frying of food. For these reasons preservatives with antioxidant activity have been added to packaged foods for many years. Major food preservatives include sorbates, benzoates, and synthetic antioxidants such as tertiary butylhydroquinone, propyl gallate, butylated hydroxytoluene (BHT) and butylated hydroxyanisole (BHA). The trends towards improving shelf life for products, away from synthetic chemicals in food and the increasing number of low fat (i.e. high moisture) convenience foods all require safe and effective preservatives. Thus there is renewed interest in the potential of "natural" and more water soluble antioxidants.

Such "biopreservatives" include vitamins C and E, which are natural antioxidants, the tocopherols and herbal extracts, particularly tea-, sage- and rosemary-based antioxidants. There is an especial need for extracts produced without the use of solvents as this is seen as being at odds with consumer concerns about solvents in general.

In the light of all these dietary and technological changes, the inherent antioxidant properties of all the plant-based polyphenols offer natural options to food manufacturers.

Some of the most commonly occuring polyphenols are the flavonoids with a large number of phenolic hydroxyl groups attached to ring structures that confer the antioxidant activity. They are multifunctional, acting as reducing agents, hydrogen donating antioxidants and singlet oxygen quenchers. They are ubiquitously distributed in the plant kingdom, being present in all vascular plants. They are responsible for the intense colours in all parts of a plant and estimated dietary intakes range from 500-1000mg/day of mixed flavonoids. This far exceeds the daily consumption of vitamin E and b -carotene. The flavanols in particular, (the monomers catechin, epicatechin and their gallate esters as well as the oligomeric proanthocyanidins) are major constituents of many fruits, grains and beverages such as green and black teas, coffee, chocolate and wine. Thus there is a long history of their presence in the human diet, at rather high levels. Recently, a burgeoning scientific literature has reported on their health benefits. This includes the fact that these constituents of red wine could be at the root of the French Paradox – the low incidence of coronary heart disease in the southern French despite their high fat diet and tendency to smoke – as well as many papers reporting the prevention of cardiovascular diseases and cancer and other degenerative diseases, through their antioxidant and radical scavenging activities.

It is therefore obvious that a natural extract of grape seeds (ACTIVIN, from Vitis vinifera), produced by an aqueous process and reasonably soluble in water, consisting almost entirely of the catechin monomers and oligomeric proanthocyanidins would be a natural candidate for a food preservative. This extract also satisfies two other basic conditions for an antioxidant. First, when present in low concentration relative to the substrate to be oxidized it can delay, retard or prevent the auto oxidation or free radical-mediated oxidation. Second, the resulting radical formed after scavenging must be stable - through intramolecular hydrogen bonding on further oxidation. In addition, many in vitro studies have defined the antioxidant potential of these polyphenols as direct radical scavengers and in some systems they show greater efficacy, on a mole for mole basis, than the antioxidant nutrients Vitamins C and E and b -carotene. ACTIVIN has other benefits as a natural antioxidant; those of safety (GRAS), low volatility and opportunity for higher use levels if required.

As we have said, the main purpose of adding antioxidants to food systems is to delay the accumulation of free radicals and thus enhance their oxidative stability. In this series of experiments we have not sought to identify the specific reaction involved in managing the oxidation. Rather, we have simply performed some classical food preservation experiments to show that, at the usage level of 200-400 ppm, the proanthocyanidins in ACTIVIN have measurable antioxidant activity. In addition we have ACTIVIN with some standard synthetic food-grade phenolic antioxidants such as BHT and BHA in a variety of model food and drink systems. These ranged from aqueous media to emulsions and oils in order to examine its activity in as many food types as possible.

Antioxidant assays performed with proanthocyanidins include DPPH radical scavenging, as well as the scavenging of hydroxyl radicals generated by Fenton’s reaction and the Rancimat inhibition of the oxidation of lard. We decided not to repeat these types of assays but use model food and drink systems to confirm ACTIVIN’s antioxidant functionality. In addition, flavonoids are known to possess vitamin C stabilizing and antioxidant-dependent vitamin C-sparing activities. We designed a short experiment to investigate whether this action can also be observed with ACTIVIN using a vitamin C-enhanced model beverage system similar to a non-carbonated, sweetened fruit drink.

Study 1 –DEGRADATION OF VITAMIN C AS AN EXAMPLE OF A TYPICAL OXIDATION REACTION IN A BEVERAGE - INVESTIGATION OF ACTIVIN AS AN ANTIOXIDANT

Introduction

Oxidation-reduction reactions are common foods. Although some are beneficial, most lead to detrimental effects including degradation of vitamins, pigments and lipids with loss of nutritional value and development of off-flavours. An example of the loss of vitamins is the exposure of a vitamin C containing drink to air, which results in the oxidization and subsequent reduction of vitamin C.

Using a simple beverage system consisting of water, sugar, acid, flavor, and Vitamin C (60 mg/l), we investigated the ability of ACTIVIN to decrease the oxidation of Vitamin C in the presence of air. This investigation was essentially an accelerated shelf-life study, carried out in uncovered vessels at 10° C.

Method

In a model non-carbonated, fruit flavored beverage, we added 60 mg/liter of Vitamin C and either of 2 concentrations of ACTIVIN: 100 and 200ppm. The samples, each of 250ml, were stored in a refrigerator in open containers to promote oxygenation (as in Patent # 5,141,758). Individual sample drinks were analysed on days 1 and 22 for ACTIVIN and twice weekly for Vitamin C. For the samples containing ACTIVIN, syrup of sugar, water and ACTIVIN was created and blended to the base formula containing water, citric acid and ascorbic acid. The sugar syrup was necessary to solubilize the ACTIVIN.

Vitamin C Analysis:

An HPLC method, developed by National Food Laboratories Inc, Dublin, CA was

utilized (see addendum 1).

The method included a standard curve of the model drink containing various

concentrations of Vitamin C.

ACTIVIN Analysis:

Fresh Samples for Calibration were prepared containing 50 ppm, 100 ppm, 150 ppm, 200 ppm and 300 ppm in the model drink containing Vitamin C .

A validated HPLC method for the quantitation of ACTIVIN has been developed by DCNI. This is Analytical Method 01-001-01 (see addendum 2).

Vitamin C Analyses :

The recovery of vitamin C for the standard curve was excellent. The standard error of the measurement (based on repetition of one sample 5 times) was 0.022. Table 1 and Figure 1 depict the change in vitamin C levels over the duration of the study.

ACTIVIN Analyses:

The recovery of ACTIVIN from the model drink was excellent (100 %) with a regression coefficient of 99.99 and a standard error of the measurement of 0.1 (see Table 2).

HPLC analysis confirmed the presence of ACTIVIN in drink samples 1 and 2 at the beginning and end of the oxidation study (days 1 and 22) and these results are depicted in Table 3. Figures 2a and 2b depict the chromatograms of these model drinks on Days 1 and 22 and confirm that ACTIVIN is not degrading in the beverage samples over the course of the study.

Vitamin C sparing activity:

The data were statistically analyzed using two techniques: analysis of variance (SAS) and an interactive modeling software (Modda). Using both techniques the samples with ACTIVIN at both concentrations were found to be statistically different from the Vitamin C alone control at the 95% Confidence Interval, indicating that the ACTIVIN was able to prevent degradation of Vitamin C. Interestingly, there was no difference between the sparing effects of either concentration of ACTIVIN.

Using interactive modeling statistics, it was found that (a) ACTIVIN is modifying the response, namely the disappearance of ascorbic acid, and that (b) there is NO interaction between time and the concentration of ACTIVIN. This confirms the independence of the Vitamin C sparing results over time. A model was generated, and although not perfect, Figure 3 mathematically describes the observations.

Figure 2a: Chromatograms of ACTIVIN content in samples of Drink #1{100 ppm ACTIVIN} at day 1 (blue) and day 22 (red).

From the results observed the addition of ACTIVIN at either 100 ppm or 200 ppm decreased the degradation of Vitamin C in the model fruit drink. This shelf life test of oxidative degradation was accelerated test because the samples were left uncovered and were stored at the relatively high temperature of 10° C. In addition, it was designed to assess broad differences in the ability of ACTIVIN to alter the degradation of Vitamin C.

A mechanism of action for the observed Vitamin C-sparing action has been proposed by Fereidoon Shahidi. He suggests that ACTIVIN phenolics such as the proanthocyanidins can regenerate ascorbic acid according to the following reactions (Nutraceutical and Functional Foods, a short course, February, 2001).

Vit C-OH + RO* Þ Vit C-O* + ROH

Introduction

*estimated concentrations based on residual weight after methanol removal

Discussion

This was short term, preliminary examination of ACTIVIN’s antioxidant activity in an oil-based media. Due to its poor solubility in oil, additional testing of grape seed extracts will be required with better carriers to improve their solubility or dispersibility in corn oil. Despite non-optimised experimental design, ACTIVIN showed significant ability to limit peroxide formation over time in stripped corn oil.

Introduction

Because of the poor solubility of ACTIVIN in corn oil in Study 2, we chose to extend the evaluation of ACTIVIN’s antioxidant potential to an oil in water emulsion, such as a salad dressing. The study design utilized the secondary measurement of lipid oxidation, namely the formation of n-hexanal, the end product of the oxidation on canola oil. Finally, this evaluation was planed to mimic a typical accelerated shelf life study routinely used in the food industry.

Method

A full fat salad dressing containing canola oil was prepared in bulk using a Waring blender. Eight batches of 3000 g were blended to make one master batch. The master batch was divided into eight lots of 3000g and the appropriate level of ACTIVIN #2, ACTIVIN #1 or BHT/BHA was added (see Table 5). The dressings were packed on ice and sent overnight to Medallion Laboratories for hexanal measurements over time. Samples were divided into 8 oz glass jars (for each pull date) and placed at 131° F.

Hexanal measurements were taken at time 0, 2, 7,and 9 days and 2, 3, 4, 6, 8 and 12 weeks. Volatiles, including n-hexanal, were separated by gas chromatography with a flame ionization detector and quantified. The dressing was also subjected to sensory analysis to determine the point of rancidity.

The Arrhenius equation was used to calculate activation energy and estimate the shelf life of the emulsion based upon the amount of lipid oxidation in each sample. The main value of the Arrhenius plot is that one can collect data at a high temperature and then extrapolate for the shelf life at a lower temperature.

Antioxidant additions to the emulsion

Results

The point of rancidity was found to be 30 ppm n-hexanal by the expert panel. Table 5 summarises the n-hexanal concentrations in the headspace above the emulsion over time. As expected, these rise from 1.6mg/Kg food at day 0 to 88.1mg/Kg food after 56 days storage at 131° F in a control emulsion containing no antioxidant.

Figure 4 is a graph of the n-hexanal generated data at 131° F. The control dressing was rancid by day 14. The emulsions containing 200 and 500ppm ACTIVIN #1 extended the date at which rancidity was reached to 42 and 28 days respectively. For ACTIVIN #2-containing samples failure occurred at 19 and 28 days respectively. For BHT/BHA containing emulsions, failure occurred at ~30days.

As expected, the sample with no antioxidant took the least number of days to reach 30ppm hexanal, and by calculation, the shortest shelf life (379 days). In contrast, the BHA/BHT mixture remained under 30ppm for 29 days, and by

calculation, had the longest shelf life (2361 days). ActiVin (200 ppm and 500 ppm) remained under 30 ppm for 42 and 29 days respectively, and by calculation had shelf lives much greater than no antioxidant added and slightly less than BHA/BHT (756-1180 days).

Shelf life estimates for an emulsion, determined by rancidity

na = not applicable (levels stayed below 30 ppm for length of study)

These three studies together indicate that ACTIVIN, a plant-derived proanthocyanidin containing extract, has significant antioxidant properties when added to both aqueous and lipid model food media. At the usage levels proposed, 200-400 ppm, ACTIVIN has been shown to have sufficient antioxidant capability to offer a natural alternative to synthetic antioxidant compounds such as BHA, BHT, PG and TBHQ.

Another advantage to the use of ACTIVIN as an antioxidant is that this functionality occurs at usage levels below those at which the inherent bitterness and astringency are perceived.