A study of the enzymatic hydrolysis of fish frames using model systems

A model system was employed to study the operating conditions and primary parameters of enzymic hydrolysis of cod proteins. Pancreatin, papain, and bromelain were used to hydrolyse minced cod fillets under controlled conditions and with the rate of hydrolysis being continually monitored via both the pH-stat and TNBS method. The two methods were compared and evaluated. The rate of protein solubilisation was plotted against the degree of hydrolysis (DH). Dry fish protein hydrolysate (FPH) powders having short, medium and high degrees of hydrolysis (DH of approximately 8%, 11% and 16% respectively) were produced and analysed for their molecular weight distribution, using size exclusion chromatography. Almost complete protein solubilisation (75 g soluble protein per kg hydrolysis solution) could be achieved within an hour, at 40°C, at 1% enzyme/substrate ratio (w/w) with papain and bromelain. The pH-stat was found capable of continuously following the rate of hydrolysis but only at low DH. The TNBS could be accurately used even at high DH to estimate the percentage of the peptide bonds cleaved, but required chemical analysis of withdrawn samples

Investigations on the emulsifying properties of egg white protein : a thesis presented in partial fulfilment of the requirements for the degree of Master of Food Technology at Massey University, Auckland, New Zealand

Figures 2.2 & 2.3 were removed for copyright reasons, but Figure 2.1 remains for ease of access.Egg white proteins (EWP) have excellent foaming and gelling functional properties. However, their emulsifying properties are considered poor when compared to soy proteins or milk proteins. Some studies have attributed the poor emulsifying properties to the hydrophobic amino acid groups buried deeply in the interior of the protein conformational structure which is crucial for emulsification. Several methods, such as heat treatment, acid/acid-heat treatment, Maillard reaction, phosphorylation and enzymatic hydrolysis, have been used by some researchers to improve the emulsifying properties of EWP. Preliminary experiments carried out in this study showed that oil-in-water (O/W) emulsions prepared with egg white liquid (EWL) generated lots of visible large aggregates, which no other study has reported. Therefore, it was important to investigate the factors responsible for the formation of these aggregates. Investigations into improving EWP's emulsifying properties could offer opportunities in developing unique and well-defined egg white-based emulsions. The objective of this research project was to produce egg white emulsions with little or no aggregates. This thesis comprises three main parts. The first part focused on the effects of pH and heat treatment on protein aggregation and partial denaturation of proteins in EWL. The second part investigated the effects of heat treatment, oil concentration and protein concentration on the reduction of large visible aggregates in emulsions prepared with EWL. The third part studied the effect of enzymatic hydrolysis on the degree of hydrolysis and emulsifying properties of EWP hydrolysates. The emulsifying properties of original EWP and EWP hydrolysates were characterised in terms of size and zeta (ζ)-potential of emulsion droplets and emulsion stability (e.g. turbidity, microscopic examination and phase separation). Firstly, an experimental study was carried out to evaluate the effect of pH on protein aggregation and precipitation in EWL containing different protein concentrations (0.5, 1, 2, 3, 4, 5 and 10% w/w). It was found that at all the protein concentrations used and at pH less than around 5, ζ-potential values were all positive but decreased as pH increased from 2 to 5. At pH 5, ζ-potential was close to zero (this is the pI of most egg white proteins), while, at pH levels above 5, ζ-potential became negative and increased as pH increased from pH 5 to 11. The spectral absorbance (turbidity) of emulsion samples was also measured at 600 nm which revealed that for all protein concentrations, turbidity was observed to be higher at acidic pH of 3, 4 and 5, indicating the aggregation of EWP. At alkaline conditions of pH 7, 8, 9 and 10 the EWL solutions remained to be transparent. The effect of heat treatment and holding time on the denaturation of EWP in EWL was also studied at different temperatures (57-62oC) and heating times (0-19 minutes). Higher turbidity due to protein aggregation was observed as temperature increased from 57 to 62oC and the heating time increased from 5 to 19 minutes. It is therefore concluded that EWL can be safely pasteurized with little or no denaturation or aggregation at around 57-58oC for less than 5 minutes. At 60oC, it was observed that EWL began to thicken and after 5 minutes coagulation and gelation occurred rapidly. Studies were also carried out to determine the cause of visible large aggregates formed in emulsions prepared with EWL using various factors, such as heat treatment, oil concentration and protein concentration. It was found that heat treatment (60oC for 30 minutes) of 1% (w/w) EWP solution prior to homogenisation had no effect on reduction of aggregates in emulsions containing 5, 10, 15 and 20% (w/w). However, the formation of aggregates was reduced significantly as oil concentration was reduced to 5%. Therefore, the effect of lower oil concentrations (1, 3, 5, 6, 7 and 10% w/w) on the formation of aggregates in emulsions prepared with 1% or 3% EWP concentrations was also investigated. Little or no visible aggregates were formed when emulsions were prepared with 1% EWP and ≤ 5% oil or 3% EWP and 1% oil. Therefore, the results indicated that both protein and oil concentrations played a significant role in the formation of visible aggregates in emulsions prepared with EWP as an emulsifier. The effect of EWP concentrations (0.1, 0.3, 0.5, 0.8, 1 and 2% w/w) on the formation and properties of 5% oil emulsions at ~pH 8 was then investigated. It was discovered that little or no aggregates were produced in emulsions when prepared at 0.1-1% EWP while large aggregates were formed at 2% EWP concentration. The size of emulsion droplets was observed to increase significantly from 242.1 to 703.7 nm as protein concentration increased from 0.1 to 2%. ζ-potential was however not significantly affected by protein concentration and ranged from -35.3 to -39.2 mV. The emulsions prepared were also heat treated at 60-90oC for 30 minutes. No sign of instability with a significant change in the size of emulsions due to heat treatment was observed from all emulsion samples prepared at different EWP concentrations (0.1 - 2%). However, phase separation of the emulsions was observed upon freezing at -20oC and thawing at 4 and 20oC, respectively, at all protein concentrations used. Also, the stability of emulsions was affected by the addition of salts, such as CaCl2 (5-100 mM) and NaCl (50-600 mM), with an increase in droplet size and phase separation. However, the emulsions were relatively more stable to salt-induced flocculation, especially against NaCl, at higher protein concentration (1-2%) than lower protein concentrations (0.1-0.8%). Lastly, the effect of pH 2-10 was also determined from the emulsions prepared at 1% EWP and 5% oil. Extensive droplet aggregation was observed at pH 4 and 5 as expected which is around the pI of most egg white proteins. On the other hand, it was not observed at extremely acidic pH 2.0 and alkaline pH 9-10 and in the control emulsion prepared at pH 8.3. In another part of the study, the effects of enzyme type (bromelain, ficin and papain), enzyme concentration (0.3, 0.5, 1, 2 and 4% w/w; enzyme/substrate (E/S) ratio) and hydrolysis time (0, 30, 60 and 120 minutes) on the degree of hydrolysis (DH) of EWP were investigated by diluting EWL containing 10% EWP to different EWP concentrations followed by adding enzymes into the EWL solutions. DH was observed to increase significantly (p < 0.05) with increasing enzyme concentration and hydrolysis time. A significant difference (p < 0.05) among the different types of enzymes was only observed from the samples with 4% E/S ratio at 120 minutes of hydrolysis time. Papain yielded the highest DH of 7.69% while bromelain and ficin yielded similar DH levels of 5.03% and 4.99%, respectively. The results of SDS-PAGE revealed that the protein bands corresponding to ovalbumin and ovotransferrin disappeared due to their enzymatic hydrolysis into smaller peptides but it was not significantly different between the samples treated with different E/S ratios and hydrolysis reaction times. The effects of enzyme concentration, DH and hydrolysis time on the emulsifying properties of hydrolysed EWP prepared with bromelain and ficin were investigated. Surprisingly, enzymatic hydrolysis significantly improved the appearance of emulsions prepared with EWL containing hydrolysed EWP by producing an emulsion free of aggregates compared to the control emulsions prepared from original EWP which had lots of large aggregates in it. For example, emulsions containing 10% oil and various EWP concentrations (1, 5 and 10%) prepared with hydrolysed EWP (4% E/S, DH 5.16%) yielded smaller droplet size (0.66-0.98 μm) than those of original EWP emulsions (1.22-39.35 μm). However, phase separation occurred immediately after preparation at all protein concentrations (1, 5 and 10%) used while phase separation occurred in only emulsions stabilised with 5 and 10% original EWP. When the emulsions were heat treated at 60-90oC for 0-30 minutes, gelation occurred in the emulsions prepared with 5 and 10% EWP concentrations while the emulsions prepared with 1% EWP had no gelation but had aggregation and phase separation after heat treatment. Emulsions prepared with 1% E/S ficin (DH 4.03% and 4.96%, respectively, after 2 and 4 hours of hydrolysis time) yielded smaller droplets size (0.75-0.87 μm) than droplet size (6.40-7.37 μm) of emulsions prepared with 1% E/S bromelain (DH 4.10% and 4.87% after 2 and 4 hours of hydrolysis time). Droplet size decreased as hydrolysis time increased from 2 to 4 hours for both ficin and bromelain hydrolysates with phase separation occurring the following day after the preparation of emulsions. Thus, DH and enzyme type had some influence on the emulsifying properties of EWP hydrolysates. In conclusion, this study demonstrated that egg white emulsions can be prepared with little or no aggregates using low oil (≤5%) and low protein (1%) concentrations and by enzymatic hydrolysis of EWP. Emulsions containing 5% oil prepared with a relatively higher protein concentration (1-2%) were more stable to destabilization to ionic strength (salt concentration), especially against NaCl. These could lead to production of egg white protein based-emulsions with distinct appearance and characteristics

In vitro and in vivo anthelmintic efficacy of plant cysteine proteinases against the rodent gastrointestinal nematode, Trichuris muris

We examined the mechanism of action and compared the anthelmintic efficacy of cysteine proteinases from papaya, pineapple, fig, kiwi fruit and Egyptian milkweed in vitro using the rodent gastrointestinal nematode Heligmosomoides polygyrus. Within a 2 h incubation period, all the cysteine proteinases, with the exception of the kiwi fruit extract, caused marked damage to the cuticle of H. polygyrus adult male and female worms, reflected in the loss of surface cuticular layers. Efficacy was comparable for both sexes of worms, was dependent on the presence of cysteine and was completely inhibited by the cysteine proteinase inhibitor, E-64. LD50 values indicated that the purified proteinases were more efficacious than the proteinases in the crude latex, with purified ficin, papain, chymopapain, Egyptian milkweed latex extract and pineapple fruit extract, containing fruit bromelain, having the most potent effect. The mechanism of action of these plant enzymes (i.e. an attack on the protective cuticle of the worm) suggests that resistance would be slow to develop in the field. The efficacy and mode of action make plant cysteine proteinases potential candidates for a novel class of anthelmintics urgently required for the treatment of humans and domestic livestock

Anthelmintic action of plant cysteine proteinases against the rodent stomach nematode, Protospirura muricola, in vitro and in vivo

Cysteine proteinases from the fruit and latex of plants, including papaya, pineapple and fig, were previously shown to have a rapid detrimental effect, in vitro, against the rodent gastrointestinal nematodes, H eligmosomoides polygyrus (which is found in the anterior small intestine) and Trichuris miti,is (which resides in the caecum). Proteinases in the crude latex of papaya also showed anthelmintic efficacy against both nematodes in vivo. In this paper, we describe the in vitro and in vivo effects of these plant extracts against the rodent nematode, Protospirua muricola, which is found in the stomach. As in earlier work, all the plant cysteine proteinases examined, with the exception of actinidain from the juice of kiwi fruit, caused rapid loss of motility and digestion of the cuticle, leading to death of the nematode in vitro. In vivo, in contrast to the efficacy against H. polygyrus and T. muris, papaya latex only showed efficacy against P. muricola adult female worms when the stomach acidity had been neutralized prior to administration of papaya latex. Therefore, collectively, our studies have demonstrated that, with the appropriate formulation, plant cysteine proteinases have efficacy against nematodes residing throughout the rodent gastrointestinal tract

Common respiratory diseases.

The self-medication phenomenon in upper respiratory tract infections, rhinosinusitis, asthma, and chronic obstructive pulmonary disease are significant and will continue to increase. Current level of evidence is poor because of the small number of good quality studies, small sample size, short duration, and variation in the composition of the herbal interventions or therapies. The current review points to several potential therapies that could be effective either alone, or as adjuncts to conventional therapies

The bracteatus pineapple genome and domestication of clonally propagated crops

Domestication of clonally propagated crops such as pineapple from South America was hypothesized to be a 'one-step operation'. We sequenced the genome of Ananas comosus var. bracteatus CB5 and assembled 513 Mb into 25 chromosomes with 29,412 genes. Comparison of the genomes of CB5, F153 and MD2 elucidated the genomic basis of fiber production, color formation, sugar accumulation and fruit maturation. We also resequenced 89 Ananas genomes. Cultivars 'Smooth Cayenne' and 'Queen' exhibited ancient and recent admixture, while 'Singapore Spanish' supported a one-step operation of domestication. We identified 25 selective sweeps, including a strong sweep containing a pair of tandemly duplicated bromelain inhibitors. Four candidate genes for self-incompatibility were linked in F153, but were not functional in self-compatible CB5. Our findings support the coexistence of sexual recombination and a one-step operation in the domestication of clonally propagated crops. This work guides the exploration of sexual and asexual domestication trajectories in other clonally propagated crops

Extraction, purification and characterisation of bromelain from pineapple crowns and its application / Nadzirah Kamarul Zaman

Pineapple crowns are an agricultural waste available in large quantity particularly from canned pineapple industry. Alternatives to its efficient utilisation are necessary as the crowns contain high enzyme activity which can be potentially used as a meat tenderiser. Thus, the aims of this study are to extract and purify bromelain from the pineapple crowns. The purified bromelain was freeze dried to produce bromelain powder which was later applied to tenderise the toughest Brahman part, round. The action of bromelain in tenderising beef was affected by different pHs of beef, immersion temperatures and times, and bromelain solution concentrations. Thus, response surface methodology (RSM) was used to determine the feasible optimum condition for the beef tenderisation by bromelain. The effect of bromelain treatment on the physico-chemical properties of beef and its nutritional quality was determined. The nutritional quality of beef was determined from the proximate extent between the bromelain-treated beef and the reference pattern protein and compared with the untreated beef. SDS-PAGE revealed that bromelain from pineapple crowns is a monomeric with a molecular weight of 30 kDa. The bromelain powder was less pure compared to that of standard bromelain powder

Design of an anti-inflammatory diet (ITIS diet) for patients with rheumatoid arthritis.

Background:Rheumatoid arthritis (RA) is a chronic systemic autoimmune disease that affects synovial joints, leading to inflammation, joint destruction, loss of function, and disability. Although recent pharmaceutical advances have improved treatment of RA, patients with RA often inquire about dietary interventions to improve RA symptoms, as they perceive rapid changes in their symptoms after consumption of certain foods. There is evidence that some ingredients have pro- or anti-inflammatory effects. In addition, recent literature has shown a link between diet and microbiome changes. Both diet and the gut microbiome are linked to circulating metabolites that may modulate inflammation. However, evidence of the effects of an anti-inflammatory and probiotic-rich diet in patients with RA is scarce. There is also a need for biological data to support its anti-inflammatory effects. Methods:The main goal of this study is to delineate the design process for a diet tailored to our RA population. To achieve this goal, we collected information on diet, supplements, cooking methods, and intake of different ingredients for each patient. Different groups were interviewed, and their feedback was assessed to design a diet that incorporates suggested anti-inflammatory ingredients in a manner that was easy for patients to adopt based on their lifestyles and backgrounds. Results:We designed a diet that includes a high intake of potential anti-inflammatory ingredients. Feedback from highly motivated patients was critical in constructing an anti-inflammatory diet (ITIS diet) with elevated adherence. Conclusion:In order to tailor our diet, we surveyed our patients on several different parameters. We obtained important feedback on how feasible our ITIS diet is for RA patients. Using this feedback, we made minor improvements and finalized the design of the ITIS diet. This diet is being used in an on-going pilot study to determine their anti-inflammatory effect in pain and joint swelling in RA patients. Trial registration:Not applicable