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Validating functional properties of foodstuffs

Published: September 4, 2007
By: FRANK R. DUNSHEA (Courtesy of Alltech Inc.)

The notion of eating foods that have beneficial effects on consumer health above those of adequate nutrition is one that sits well with today’s consumers. Consequently the development of ‘functional foods’ and their impact on our health has received much attention over the last decade from scientists, health professionals, health policy makers, primary and food producers.

The majority of the functional foods on the market today aim to offset the incidence of a number of major human non-communicable diseases, with the main constituents of the functional food products being dietary fibre, probiotic cultures, minerals and vitamins, oligosaccharides, unsaturated fatty acids and omega fatty acids.

Functional foods research within our research group has involved using the pig as a biomedical model to assess the functional attributes of food and horticultural products, increasing functional attributes of animal products such as milk and meat and increasing the functional attributes of processed products through inclusion of healthpromoting compounds. In this paper I would like to discuss some of our activities in these areas.


Animal models to assess functional properties

BOWEL HEALTH

The pig is a good animal model for human nutrition, because of the many similarities between pigs and man in the anatomy and physiology of the digestive (Book and Bustad, 1974; Swenson, 1977) and cardiovascular (Martin, 1964; Lumb, 1966) systems.

Furthermore, the advantage of the pig in biomedical research is the similarity in the major lipoprotein subclass of LDL physico-chemical characteristics, albeit that the levels of plasma lipids are lower in pigs than human subjects (Chapman and Goldstein, 1976).

The first studies that we conducted using the pig as a model for the human were aimed at investigating the interactions between dietary fibre and resistant starch and their effects on the risk factors for colon cancer and bowel health (Govers et al., 1999). By feeding pigs their respective diets and then slaughtering them in the pilot abattoir, it is possible to recover digesta from various segments of the colon (Dunshea et al., 2001) and look at risk factors for colon disease.

Sloughing of mucosal cells invalidates this technique for studies on protein digestion but it is perfectly appropriate for studies on carbohydrate and fibre digestion (Prawirodigdo et al., 1998).

In these studies we found that the combination of both resistant starch and fibre resulted in greater fermentation in the hindgut than either source alone, higher levels of butyrate (Figure 1), lower pH and concentrations of ammonia in the distal colon, the site of greatest cancer risk. Thus, dietary fibre and resistant starch had additive (and synergistic) effects on improving bowel health and reducing the risk factors for colon cancer and other disease.

Data from this study in pigs were used to develop a clinical study that has since been conducted in humans (Muir et al., 2004). A number of other workers have also found the pig to be an excellent model for investigating dietary means of manipulating colon health and as a screen before conducting clinical studies in humans (Bird et al., 2004).


FUNCTIONAL PROPERTIES OF ONIONS

Plants from the genus Allium, particularly onions (A. cepa) and garlic (A. sativum), have been consumed for their putative nutritional and health benefits for centuries. Although the health functionality of garlic has been reported extensively, very little is known about the specific benefits of onions.

Epidemiological studies have shown a correlation between diets rich in onion and reduced risk of stomach cancer in human subjects (You et al., 1989), as well as an inverse relationship with mortality from coronary heart disease (CHD) in man (Hertog et al., 1993a,b).

Compounds that have been implicated in providing a number of health-promoting attributes of onion include flavonoids, particularly the flavonol quercetin and the organosulfur compounds such as cysteine sulfoxides (CSO) (Price and Rhodes, 1997).

Sulphur compounds from garlic, and common to onion, have been shown to reduce plasma total cholesterol, LDLcholesterol and triacylglycerol (TG) levels in human subjects and rodents (Bordia and Verma, 1980; Chi, 1982; Chi and Stewart, 1982; Qureshi et al., 1983a,b; Slowing et al., 2001) while flavonols such as quercetin have also been shown to have lipid-modulating properties (Bok et al., 2002; Glasser et al., 2002).

We have conducted a number of studies in pigs to demonstrate some of these health active properties of onions.

Postprandial TG concentrations in pigs fed pungent onions were approximately 23% lower than in control pigs or the pigs fed mild onions, while fasting measurements were not significantly affected by onion consumption (Ostrowska et al., 2004; Gabler et al., 2005). Postprandial measurements of TG levels are useful, as they might reveal a state of fat intolerance that cannot be detected by the simple measurement of fasting plasma triglycerides (Karpe, 1999).

Therefore, these results suggest that the consumption of Destiny onions, at both doses investigated, resulted in beneficial changes in plasma postprandial TG concentrations. This may lead to reduced risk of CHD and atherogenesis (Zilversmit, 1979; Patsch et al., 1992).

Onion consumption resulted in a 9% lower plasma total cholesterol concentration (mean fasting and postprandial measurements) compared with the control group (Ostrowska et al., 2004). The HDL-cholesterol fractions were reduced in response to onion supplementation; however, the LDL:HDL ratio was unaffected. This suggests that the removal of cholesterol from circulation was not enhanced, and it is likely that the cholesterol-lowering action of onions was due to a reduction in cholesterol synthesis.

Also, dietary onions provided protection against copper–induced lipoprotein oxidation (Gabler et al., 2005). While cooking onions decreases their flavonol content, much of the antioxidant capacity of dietary onions is still retained after frying or steaming (Gabler et al., 2003).


FUNCTIONAL PROPERTIES OF OLIVE OIL

Epidemiological studies indicate that in Mediterranean countries there is significantly longer life expectancy and lower incidence of chronic disease such as cardiovascular disease and cancer despite high dietary fat consumption (Roche et al., 1998). These findings have been attributed to high dietary olive oil consumption, which is high in monounsaturated fatty acids (MUFA).

Olive oil also contains at least 30 phenolic compounds (Tuck and Hayball, 2002) and it is unclear whether the higher MUFA content of the olive oil or the polyphenol content contributes to the general health of Mediterranean populations. Another major health issue is osteoporosis; this bone degenerative disease state is associated with an aging population, especially among women.

Dietary lipids play an important part in bone health through both cartilage mineralisation and mediating cellular signalling pathways. Indeed, olive oil supplemented diets have been shown to enhance phosphorus absorption, retention and bone mineral density (BMD).

Therefore, we conducted a study to compare the effects of MUFA and polyphenols in olive oil on risk factors for CVD and bone density using the growing pig as a model (Ostrowska et al., 2005).

Pigs were fed diets that consisted of a wheat-based basal diet supplemented with either 12% tallow and 7% sunflower oil (TSO), 12% tallow and 7% extra virgin olive oil (TEVO), 19% extra virgin olive oil (EVO) or 19% of refined olive oil (RO) for eight weeks.

The two sources of olive oil had similar fatty acid profiles but the EVO contained twice the phenolic compounds (335 vs 150 ppm caffeic acid units) and less oxidised products (5 vs 9 mEq O2/kg) than the RO. Within our laboratory we have access to a dual energy X-ray absorptiometer (DXA) allowing us to measure serial changes in body composition and bone density (Suster et al., 2003).

In this study both fasting and non-fasting plasma TG concentrations were lower (P=0.003) in pigs fed MUFA-rich diets, while the plasma cholesterol profile was not significantly different between the treatments. The results from in vitro copper-induced lipid peroxidation, expressed in terms of conjugated dienes, showed that LDL particles in postprandial serum from pigs fed EVO diets were moderately more resistant to oxidative modification.

Daily gain, feed intake and lean and fat deposition were not significantly different between the treatments. However, the daily change in bone mass density was higher in pigs fed diets containing olive oil (1.2 vs 2.5, 6.3 and 5.2 mg/cm2 per day for TSO, TEVO, EVO and RO diets respectively, P<0.05). These data demonstrate that both extra virgin and refined olive oils attenuate postprandial hypertriglyceridemia, improve oxidation susceptibility and increase bone mineral density in growing pigs.


Validating functional properties of foodstuffs - Image 1

Figure 1. Effect of dietary fibre and resistant starch (RS) on butyrate concentrations in various segments of the colon.



FUNCTIONAL PROPERTIES OF WHEY AND OTHER DIETARY PROTEINS

Milk contains a mixture of proteins, each having unique attributes for nutritional, biological, and human food ingredient applications (Smithers et al., 1996). The major proteins present in milk include ß-lactalbumin, α-lactoglobulin, immunoglobulin, bovine serum albumin, and the caseins: κ-casein, ß-casein, and the α-caseins (Etzel, 2004). In addition, whey contains glycomacropeptide (GMP) that is cleaved from κ-casein by chymosin to initiate precipitation of the caseins, forming curd.

When casein is removed from whole milk, liquid whey remains, and a number of different proprietary processes exist to further treat or purify whey protein resulting in various whey protein isolates, some of which may be rich in specific bioactive peptides such as GMP.

Recently, there has been evidence to support a role for dietary proteins in the regulation of food intake and weight maintenance (Anderson and Moore, 2004). In addition, some protein sources contain specific peptides that may elicit a direct effect upon satiety. For example, GMP is thought to have a satiating effect and there are whey protein isolates (WPI) that are relatively high in GMP.

Therefore, we conducted a study to determine if high protein diets, including a WPI rich in GMP, have any effect on feed intake, body weight and other indices of obesity in minipigs. The minipig is an excellent model for obesity as it contains 50% body fat and displays insulin resistance (Dunshea et al., 2005).

Adult female minipigs (133 kg, 50% body fat) were randomly allocated to a 2x2 factorial design with the respective factors being source of protein (WPI, 30% GMP) vs. soy protein isolate (SPI) or level of dietary protein (11 (LP) vs. 22% (HP) CP).

After consuming their respective diets ad libitum for 10 weeks, the pigs were infused i.v. with insulin at 0.6 and 6.0 mU/(kg/min) and blood glucose and amino acids clamped at pre-infusion values by simultaneous infusions of dextrose (50% w/v) and a parenteral amino acid mix (10% w/v), respectively. Again, serial measurements of body composition were made using DXA.

Feed intake was lower in pigs fed the HP diet (2070 vs. 2352 g/ d, P<0.001), particularly in pigs fed WPI (1951 vs. 2408 g/d) as indicated by an interaction (P=0.027) between source and level.

Pigs consuming the HP diet deposited less weight (231 vs. 382 g/d, P=0.045) and had a lower ratio of fat:lean in the ham (0.70 vs. 0.76, P=0.026) at 8 weeks than those fed the LP diet. Protein source had no effect on the amount of dextrose infused to maintain euglycemia (108 vs. 115 mL/h P=0.59) but the amount infused was lower in the minipigs fed the LP diet (101 vs. 125 mL/h, P=0.048). The amount of dextrose required to maintain glycemia was higher at the higher dose of insulin (114 vs. 226 mL/h, P<0.001).

Protein source had no effect on the amino acid infusion rate required to maintain plasma lysine concentrations (50 vs. 50 mL/h, P=0.98) but the amount infused was lower in pigs fed the LP diet (45 vs. 55 mL/h, P=0.030). The amino acid infusion rate was higher at the higher dose of insulin (47 vs. 103 mL/h, P<0.001).

These data showed that a HP diet reduced feed intake, weight gain and fat deposition and reduced insulin resistance in obese minipigs. The HP diet containing WPI that was enriched in GMP had the greatest effect upon feed intake and weight gain.


BIOAVAILABILITY AND BIOEFFICACY OF DIFFERENT FORMS OF SELENIUM

Due to the relatively low level of selenium (Se) in Australian and New Zealand soils many primary products from these countries have relatively low Se levels. While it is generally accepted that selenium intakes of Australian and New Zealand consumers are sufficient to ensure no overt signs of deficiency, there is a feeling that the relatively low intakes may contribute to elevated risk for some cancers (e.g., bowel) (Clark et al., 1996).

However, selenium supplementation is problematic, as high selenium intake can be toxic, particularly if the source is inorganic (Kim and Mahan, 2001). Protein-bound selenium is more bioactive and less toxic than inorganic forms and there is interest in delivering selenium in organic forms in food products we consume.

The aim of this study was to determine the relative bioavailabilities of selenium from the Se-enriched whole milk and milk replacers supplemented with exogenous selenate or Sel-Plex® selenium yeast in neonatal pigs.

Milk enriched with selenium (1070 μg Se/kg DM; HSeM) was obtained from cows fed a supplemental diet containing Sel-Plex® (Alltech Inc.) while the control diet (135 μg Se/kg DM; LSeM) was made using milk powder from non-supplemented cows.

Additional diets were formulated by adding Sel-Plex® (HSP) or selenate (HSN) to the LSeM diet to give selenium concentrations of 1070 μg Se/kg DM. Neonatal pigs (n=54) at two days of age were trained to drink milk and after a further three days, were randomly allocated to their four respective diets (1.7 MJ/kg BW) and slaughter times (0, 7, 14, 28 and 42 days of feeding).

In pigs receiving HSeM there was a rapid increase in plasma selenium over the first 7 days after which there was a gradual increase for a further 14 days and then plasma selenium was maintained 3-fold higher than in the LSeM pigs. Plasma selenium concentrations in the HSP and HSN pigs increased by 20-25% over the first 14 days and then plateaued. Between 14 and 42 days plasma selenium levels were 45, 125, 122 and 170 μg/L for LSeM, HSN, HSP and HSeM pigs, respectively (P<0.001). Muscle selenium reached a plateau after 28 days of feeding (Figure 2). Between 28 and 42 days, muscle Se levels were 47, 106, 237 and 486 μg/kg for LSeM, HSN, HSP and HSeM pigs, respectively (P<0.001).

These data suggest that selenium in milk from cows fed Sel- Plex® is highly bioavailable and may offer a means of delivering supplemental selenium to humans.

The growing interest in the selenium content of dairy products has to some extent been fuelled by the work of McIntosh and colleagues working with casein processed from milk obtained from cows that had been supplemented with yeast-bound selenium (Sel-Plex®) (McIntosh et al., 2004).

Using an azoxymethene (AOM)-induced colon cancer model these workers found significant reductions in colonic tumour incidence (rats with tumours) and burden (tumours/rat) in rats consuming selenised casein (1 ppm Se) relative to control and yeast selenium treatments. There was an effective reduction in the benign and malignant tumours in the selenised casein group relative to control and yeast selenium treatments (McIntosh et al., 2004).
Therefore, it is possible that delivery of selenium via enriched milk and dairy products may be a means of providing health benefits to people at risk of colon or other cancers. This may be due to an additive effect of selenium and other components in dairy protein that have been found to protect against cancer in rodent models (McIntosh et al., 1995).

Within the Centre of Excellence for Functional Foods we are working with the AOM colon cancer model to determine the mode of anti-carcinogenic action of dairy bound selenium derived from cows supplemented with Sel-Plex®. We have established RT-PCR assays to determine the level of expression of key selenoproteins or proteins implicated in being either causative or preventive of cancer (Figure 3).

Further studies will make use of gene chip technologies.


Validating functional properties of foodstuffs - Image 2

Figure 2. Effects of time of feeding different forms of selenium on muscle selenium concentrations in artificially reared neonatal pigs (HSeM = high Se milk from cows fed Sel-Plex®; LSeM = milk from unsupplemented cows).



Validating functional properties of foodstuffs - Image 3

Figure 3. Rat selenoproteins and colon cancer biomarkers amplified using RT-PCR.

Conclusion

The pig is an excellent animal model for human nutrition, because of the many similarities between pigs and man in the anatomy and physiology of the digestive and cardiovascular systems.

In addition, there are obese lines of pigs that are particularly useful when studying obesity and related diseases.

In this paper I have outlined how we have used animal models, particularly the pig, to demonstrate the bioactivity and bioefficacy of a number of health active compounds in foods.

Selection of the correct biomarkers for a particular disease and new technologies such as RT-PCR and, in some cases, gene chips will allow further identification of bioactives, perhaps prior to clinical studies in humans.


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Author: FRANK R. DUNSHEA
National Centre of Excellence in Functional Foods, Department of Primary Industries, Werribee, Victoria, Australia
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