Functional properties of Australian blue lupin (Lupinus angustifolius) protein and biological activities of protein hydrolysates

Lupin is an undervalued legume despite its high protein content with known health benefits. In this research, Australian blue lupin protein was isolated and hydrolysed enzymatically to produce bioactive peptides with a view to assess their potential for nutraceutical and therapeutic applications. Pepsin, pancreatin and flavourzyme were used to enzymatically hydrolyse blue lupin protein, and the hydrolysates were subjected to molecular weight cut‐off (MWCO) fractionation. Measurement of biological activities led to the identification of angiotensin converting enzyme (ACE) inhibitory fractions in the molecular weight range of 2–3 and 3–5 kDa. For the most active fractions in this range, the ACE inhibitory activities were very significant with IC50 values from 450 to 600 μg/ml. Blue lupin protein‐derived MWCO fractions were significantly active against Gram‐positive bacteria and only a little inhibition was observed against Gram‐negative bacteria. Pancreatin hydrolysed fractions showed the best antimicrobial activities with several fractions exhibiting ≥85% inhibition against Bacillus cereus and Staphylococcus aureus. These properties reveal the potential of lupin protein hydrolysates for developing antihypertensive and host defence agents. In order to demonstrate the potential of isolated blue lupin protein in food industry, functional properties including water and oil absorption capacity, gelling properties, solubility and emulsifying properties were evaluated and found to be extremely suitable for developing functional foods with enhanced health benefits

Encapsulated folic acid in Cheddar cheese reduce methionine-induced hyper-homocysteinemia in male BALB/c mice

Methionine is the precursor of homocysteine. Elevated plasma homocysteine (hyperhomocysteinemia) is associated with occlusive vascular disease. Nutritional interventions are thought to be an effective and safe means of reducing the associated risk of disease through dietary intake of folates. We compared the impact of free and encapsulated (alginate-pectin) folic acid on methionineinduced hyperhomocysteinemia in male Balb/c mice. Cheddar cheese was used as the food carrier (folic acid incorporated into Cheddar cheese either in free form or as encapsulated capsules) and the evaluation was conducted over a 12-wk period. Mice were fed six experimental diets: (i) Met 10 [L-methionine (10 g/Kg) only intake], (ii) Met 20F [L-methionine ( 20 g/kg) with free folic acid (2 mg/kg)], (iii) Met 20E [L-methionine (20 g/kg) with encapsulated folic acid (2 mg/kg)], (iv) Met 10F [L-methionine (10 g/kg) with free folic acid (2 mg/kg)], (v) Met 10E [L-methionine (10 g/kg) with encapsulated folic acid (2mg/kg)], (vi) Control (Cheddar cheese without added folic acid). Supplementation of the diet with Met 10 and Met 20F increased plasma homocysteine to 1.8 and 6.1 times the control, respectively. The hyperhomocysteinemia caused by the dietary addition of Met 10 was counteracted by the encapsulated folic acid (Met 10E) while free folic acid (Met 10F) showed a higher result. Similarly, encapsulated folic acid caused substantial reduction in plasma homocysteine in mice fed with Met 20E compared to Met 20F. The results show that Cheddar cheese incorporated with encapsulated folates can be used as a functional food vehicle to reduce hyperhomocysteinemia

Gene expression profile in the small intestine of mice fed Lactobacillus acidophilus LAFTI L10

The effects of Lactobacillus acidophilus LAFTI L10 on gene expression in the small intestine of mice was evaluated using microarrays. Male BALB/c mice were orally fed with 108 colony forming units of L. acidophilus in skimmed milk powder for 14 days. Control mice received skimmed milk powder without L. acidophilus. After 14 days, distal end of the small intestine was excised for microarray analysis of gene expression. L. acidophilus-fed mice altered the expression of genes such as CD40 ligand, CD200 receptor-3 and trefoil factor 2, which are involved in host defence mechanisms. Overall, the expression of functional genes influenced by L. acidophilus in the small intestine of mice offer as a basis for further investigation into its probiotic effects

Survival and release of probiotic bacteria from chitosan-coated alginate-starch capsules in mice gastro-intestinal tract and effect on faecal flora and immune parameters in mice

Chitosan-coated alginate-starch (CCAS) capsules containing approximately 108 CFU of Lactobacillus casei Shirota strain cells (LCS) were fed to mice and the release profile of bacteria from CCAS capsules in murine GI tract was investigated at different time intervals. There was a complete release (108 CFU) of LCS from capsules within 12 h in mice ileal region. There was only a partial release of encapsulated LCS, in duodenal, jejunal and colon regions, while there was no release of encapsulated LCS in stomach from the CCAS capsules even after 24 h. The effect of feeding (14 days) CCAS encapsulated LCS on faecal microbial population and immune parameters in mice were also determined. The numbers of LCS recovered from the faeces confirmed adequate percentage of LCS survived passage through gastrointestinal tract. A concomitant increase in the LCS and total lactobacilli count during the test period was observed in the group fed with CCAS encapsulated bacteria compared to the one fed with free non-encapsulated bacteria. This experiment also showed that the CCAS microencapsulated LCS did not affect or mask the probiotic bacteria’s immunomodulatory activity. The effect on the specific immune response was assessed by measuring cytokine production of the mouse splenocytes. There was significant increase (p<0.05) in interleukin (IL-10) and interferon (IFN)-ʮproduction between groups fed with CCAS encapsulated LCS and free LCS compared to the control and the one fed with empty CCAS alginate capsules. Chitosan coated alginate-starch capsules can be effectively used to deliver viable bacteria safely to animal intestine for increased immunomodulatory activity

Effect of encapsulation on the survival of probiotic bacteria in the presence of starter and non-starter lactic acid bacteria in Cheddar cheese over a 6-month ripening period

Probiotic bacteria (Lactobacillus acidophilus LAFTI L10 and Bifidobacterium lactis LAFTI B94) were encapsulated in calcium-alginate hydro-gel to study the effect of encapsulation on their survival in Cheddar cheese. Twelve batches of Cheddar cheese were manufactured incorporating encapsulated and free probiotic bacteria. The survival and the effect of encapsulated probiotic bacteria on the growth of starter lactic acid bacteria (SLAB) and non-starter lactic acid bacteria (NSLAB) were assessed over a six-month ripening period. The survival of encapsulated bacteria (107 cfu/g) was found to be significantly (P<0.05) greater than that of free bacteria (105 cfu/g) at the end of six-months. Also, addition of encapsulated probiotic bacteria did not change the population of SLAB and NSLAB. This study therefore demonstrates that encapsulated probiotic bacteria survive better than free probiotic bacteria in Cheddar cheese during the long ripening period and had no effect on the SLAB and NSLAB growth during ripening

Comparison of functional assay and microarray analysis for determination of Lactobacillus acidophilus LAFTI L10 induced gut immune responses in mice

Probiotics are emerging as functional food supplements that are known to promote specific health benefits beyond basic nutritional functions. The current study evaluated the ability of Lactobacillus acidophilus LAFTI L10 in modulating gut immune responses in mice. Mice were orally fed with L. acidophilus for 14 days, during which they were immunized twice with 10 µg of cholera toxin (CT). The immunoglobulin (Ig)-A response to CT in intestinal fluid and serum were significantly enhanced by L. acidophilus. Mice fed with L. acidophilus increased the number of interferon (IFN)-γ, interleukin (IL)-6 and IL-10 producing cells in the small intestine. Microarray analysis is known to be more sensitive and predictive than functional assays. For this reason, gene expression analysis was chosen as an alternative approach in determining the immunomodulatory effects of L. acidophilus. Gene expression profiles in small intestine of mice fed with L. acidophilus did not uncover the effects related to immune functions that were detected through functional assays. Therefore, functional assays are superior in predicting the immunomodulatory responses of probiotic bacteria

Impact of alginate-chitosan encapsulated flavourzyme on peptide and amino acid profiles in Cheddar cheese

The effect of alginate-chitosan encapsulated Flavourzyme on the water-soluble and water-insoluble peptides and amino acids released during Cheddar cheese ripening was investigated. Increased and prolonged proteolysis was observed in cheeses incorporated with encapsulated Flavourzyme. Rapid proteolysis and increased accumulation of hydrophobic or high molecular weight peptides was however observed in cheeses with free Flavourzyme compared to control (without Flavourzyme) or cheeses with encapsulated Flavourzyme. Concentration of watersoluble peptides increased with the concentration of encapsulated Flavourzyme in the cheese. Most free amino acids were about 3 times greater in cheese with encapsulated Flavourzyme compared to control cheese after 30 days ripening and about 7 times greater after 90 days ripening. Total amino acid content was highest in cheese with encapsulated Flavourzyme followed by free Flavourzyme treated cheese and lowest in control cheese. Flavourzyme encapsulated in slow release alginate-chitosan matrix can be a potential delivery system for flavour enhancement during cheese ripening

Identification of bioactive peptides in commercial Australian organic cheddar cheeses

This study examined two commercial Australian organic cheddar cheeses (A and B) for the presence of antimicrobial, antioxidant and antihypertensive activities. The antimicrobial activity of the peptides was determined by the growth inhibition of Escherichia coli, Bacillus cereus and Staphylococcus aureus. The bacteria were inhibited the greatest by the whole peptide extracts derived from organic cheddar cheese A. Overall, B. cereus was inhibited the most by fractionated peptides derived from organic cheddar cheese A between >5 kDa and 0.05) were observed. Antihypertensive activity was determined by inhibition of the angiotensin I-converting enzyme. Overall, organic cheddar cheese B peptides less than 5 kDa had the lowest IC50 value (0.114 mg/mL), followed closely by the fraction containing peptides between 5 kDa and 10 kDa from organic cheddar cheese B (0.122 mg/mL). This study has shown that peptides derived from Australian organic cheddar cheeses potentially have antioxidant, antihypertensive and antioxidant properties

Identification of bioactive peptides in commercial Cheddar cheese

This study examined the presence of antimicrobial, antioxidant and antihypertensive peptides in three commercially available Australian Cheddar cheeses. Peptide extracts as well as fractionated peptide extracts were examined. Commercial cheese A peptides exhibited the greatest inhibition against Bacillus cereus and also commercial cheese A fractionated peptides greater than 10 kDa showed the highest inhibition against B. cereus. Commercial cheese A peptides also showed the highest inhibition of 2,2-diphenyl- 1-picrylhydrazyl (DPPH), a free radical used to measure antioxidant activity. All cheese fractionated peptides greater than 10 kDa demonstrated higher inhibition of DPPH after fractionation. Antihypertensive peptides were determined by inhibition of the angiotensin-converting enzyme (ACE). Overall, commercial cheese A had the lowest concentration required to inhibit ACE and commercial cheese A fractionated peptides lower than 5 kDa had the lowest inhibition after fractionation. These preliminary findings suggest that peptide extracts of three commercial Australian Cheddar cheeses exhibit antimicrobial, antihypertensive and antioxidant properties

Isolation and characterization of nutraceutically potential ACE-inhibitory peptides from leatherjacket (Meuchenia sp.) protein hydrolysates

Bioactive peptides, especially ACE-inhibitory peptides (ACEIPs), have been shown to possess promising application as nutraceuticals. In this study, soluble and insoluble fish protein substrates from leatherjacket (Meuchenia sp.) were hydrolyzed with papain, bromelain and Flavourzyme, and the hydrolysates were used in a systematic screening for ACEIPs. Fractionation of selected <5 kDa hydrolysates obtained from insoluble papain at 6 h (LPI6h), insoluble bromelain at 2 h (LBI2h), insoluble bromelain at 8 h (LBI8h) and insoluble Flavourzyme at 2 h (LFI2h) gave five active fractions (LPI5, LPI6, LBI2, LBI5, and LFI5). The primary structures of LPI5, LPI6, LBI5, LBI12 and LFI5 fractions are EPLYV, DPHI, AER, EQIDNLQ and WDDME, having IC50 values of 0.05, 0.02, 0.11, 0.24 and 0.01 g/L, respectively. Based on the results of simulated gastrointestinal enzyme degradation and ACE inhibitory activity, the <5Ka fractions of LPI6h, LBI2h and LFI2h have potential to be used in the preparation of functional foods