Ivar Storrø

Influence of complex nutrients, temperature and pH on bacteriocin production by Lactobacillus sakei CCUG 42687.

Abstract The effects of process conditions and growth kinetics on the production of the bacteriocin sakacin P by Lactobacillus sakei CCUG 42687 have been studied in pH-controlled fermentations. The fermentations could be divided into phases based on the growth kinetics, phase one being a short period of exponential growth, and three subsequent ones being phases of with decreasing specific growth rate. Sakacin P production was maximal at 20 °C. At higher temperatures (25–30 °C) the production ceased at lower cell masses, when less glucose was consumed, resulting in much lower sakacin P concentrations. With similar media and pH, the maximum sakacin P concentration at 20 °C was seven times higher than that at 30 °C. The growth rate increased with increasing concentrations of yeast extract, and the maximum concentration and specific production rate of sakacin P increased concomitantly. Increasing tryptone concentrations also had a positive influence upon sakacin P production, though the effect was significantly lower than that of yeast extract. The maximum sakacin P concentration obtained in this study was 20.5 mg l−1. On the basis of the growth and production kinetics, possible metabolic regulation of bacteriocin synthesis is discussed, e.g. the effects of availability of essential amino acids, other nutrients, and energy.

Bitterness in Fish Protein Hydrolysates and Methods for Removal

Abstract Enzymatic hydrolysis is a processing method for recovering protein from under utilized fish biomass and fish by-products. However, the hydrolysis process often creates bitter taste in the product. The bitterness restricts the practical uses of these hydrolysates. The presence of bile in whole fish and fish viscera is shown to cause bitterness in fish protein hydrolysates. The fat and ash content could also cause bitter taste. The content of total amino acids and hydrophobic amino acids did not correlate with bitterness. Three different methods were used to eliminate or reduce bitterness from FPHs after enzymatic hydrolysis with commercial enzymes: (1) treatment with endopeptidases (Flavourzyme(®)), (2) extraction with butanol and (3) treatment with cholestyramine resin. Flavourzyme(®) did not reduce the bitterness. The use of butanol and cholestyramine resin separately or in combination reduced the bitter taste from FPH to levels barely discernible by our sensory panel in 1% concentration.

Uptake of lactose and continuous lactic acid fermentation by entrapped non-growing Lactobacillus helveticus in whey permeate

Abstract Continuous production of lactic acid from lactose has been carried out in a stirred-tank reactor with non-growing Lactobacillus helveticus entrapped in calcium alginate beads. A considerably longer operation half-life was obtained in a continuously operated reactor than in a batch-operated reactor. It is possible to simulate the action of entrapped non-growing cells on the basis of information from diffusion and kinetic experiments with suspended free cells. The simulation fit the experimental data over a broad range of substrate concentrations if the specific lactic acid production rate, qP, was used as a variable parameter in the model. The dynamic mathematical model used is divided into three parts: the reactor model, which describes the mass balance in a continuously operated stirred-tank reactor with immobilized biomass, the mass-transfer model including both external diffusion and internal mass transfer, and the kinetic model for uptake of substrate on the basis of a Michaelis-Menten-type mechanism. From kinetic data obtained for free biomass experiments it was found, with the use of non-linear parameter estimation techniques, that the conversion rate of lactose by L. helveticus followed a Michaelis-Menten-type mechanism with KS at half-saturation=0.22±0.01 g/l. The maximum specific lactose uptake rate for growing cells, qS,max, varied between 4.32±0.02 g lactose g cells-1 h-1 and 4.89 ±0.02 g lactose g cells-1 h-1. The initial specific lactose uptake rate for non-growing cells, qS,0, was found to be approximately 40% of the maximum specific lactose uptake rate for growing cells.

Influence of human gastric juice on oxidation of marine lipids--in vitro study.

This study evaluates whether marine lipids can oxidise in acidic stomach environment and whether authentic gastric juice has the potential to act as a pro- or anti-oxidative medium. Oxidation of herring lipids in emulsions and liposomes was followed in in vitro digestion models containing authentic human gastric juice, and compared to models containing hydrochloric acid solution. Peroxide value, concentration of thiobarbituric acid reactive substances and oxygen uptake rate increased in all the models during 2.5 h incubation at pH 4 and 37 °C in darkness. The markers showed no difference between oxidation in gastric juice and hydrochloric acid solution. Gastric juice reduced the prooxidant activity of iron ions measured as oxygen uptake rate, but did not reduce the activity of methemoglobin. Berry juice, green tea, red wine, and caffeic acid reduced oxygen uptake in the acidic environments while coffee, ascorbic acid and orange juice increased oxidation. Beverages accompanying foods containing marine lipids will therefore affect the course of post-prandial lipid oxidation.

Hydrolysis of Cod (Gadus morhud) By-Products

Abstract The main aim of hydrolysis of by-products is to obtain the highest possible amount of valuable components. Backbones and liver from farmed cod (Gadus morhua)were hydrolyzed and effect of different factors such as initial inactivation of endogenous enzymes, dilution prior to hydrolysis and centrifugation conditions on the yield and functional properties of the different fractions was evaluated. Samples with active endogenous enzymes gave significantly higher dry yields of emulsion, fish protein hydrolysate (FPH) and reduced dry yield of sludge. In the presence of both active endogenous and commercial enzymes, yield of dried FPH was higher than yield of dried sludge. Yield of protein containing fractions was more dependent on the initial heating of the system than on amount of added water. Centrifugation forces were important for separation of oily fractions, but not for separation of protein fractions. The oil and emulsion fractions consisted mainly of triglycer-ides, while phospholipids were found mostly in the protein containing fractions. The insoluble sludge contained a high amount of lipids (up to 30%) including a high content of phospholipids (up to 46%).

Kinetic studies of lipid oxidation induced by hemoglobin measured by consumption of dissolved oxygen in a liposome model system.

The effect of hemoglobin (Hb) and lipid concentration, pH, temperature, and different antioxidants on heme-mediated lipid oxidation of liposomes from marine phospholipids was studied. The rate of lipid oxidation was measured by consumption of dissolved oxygen. Heme-mediated lipid oxidation at different Hb and lipid concentrations was modeled by Michaelis-Menten kinetics. The maximum rate (V(max)) for the reaction with cod and bovine Hb as a pro-oxidant was 66.2 +/- 3.4 and 56.6 +/- 3.4 microM/min, respectively. The Michaelis-Menten constant (K(m)) for the reaction with cod and bovine Hb was 0.67 +/- 0.09 and 1.2 +/- 0.2 microM, respectively. V(max) for the relationship between the oxygen uptake rate and lipid concentration was 43.2 +/- 1.5 microM/min, while the K(m) was 0.93 +/- 0.14 mg/mL. The effect of the temperature followed Arrhenius kinetics, and there was no significant difference in activation energy between cod and bovine Hb. The rate of lipid oxidation induced by bovine Hb was highest around pH 6. Ethylenediaminetetraacetic acid (EDTA) had no significant effect on heme-mediated lipid oxidation, but alpha-tocopherol and astaxanthin worked well as antioxidants. Kinetic differences were found between iron and Hb as pro-oxidants, and the efficacy of the antioxidants depended upon the pro-oxidant in the system.

Production of High Quality Fish Oil by Thermal Treatment and Enzymatic Protein Hydrolysis from Fresh Norwegian Spring Spawning Herring By-Products

There is an increasing demand for omega-3 containing fish oils. By-products from fish fillet production can be utilized for the production of fish oils and be a valuable source of omega-3 for human consumption. The aim of this work was to evaluate industrial processes for production of quality oil for human consumption made from Norwegian spring spawning herring by-products. A mobile production plant was used to compare two industrial processes, thermal treatment (wet rendering) and enzymatic protein hydrolysis, for production of oil from herring by-products immediately after filleting. Results show that high quality herring oil can be produced from fresh by-products. The use of by-products immediately after filleting resulted in a low amount of free fatty acids for all the produced oils (below 0.4%). Thermal treatment at 70°C resulted in an oil with lower oxidation status and higher stability compared to the oils produced by enzymatic protein hydrolysis. Nevertheless, both processing methods gave a crude oil of high quality compared to crude oils on the market.

The effect of dietary antioxidants on iron-mediated lipid peroxidation in marine emulsions studied by measurement of dissolved oxygen consumption

Long chain omega-3 polyunsaturated fatty acids (LC omega-3 PUFA) are vital for physiological functions and have therapeutic and health benefits. The consumption of LC PUFA in the Western world has been below recommended intake levels the past decades, despite promotion of seafood and omega-3 supplements. Incorporation of the LC PUFA into processed food consumed on a daily basis might therefore bridge the gap between the recommended and actual consumption of LC omega-3 PUFA. Unfortunately, the development of omega-3 enriched food is hampered by a very high susceptibility of LC PUFA to oxidative deterioration. Furthermore, oxidised lipids are believed to create health risks. It has also been suggested that gastric juice may deteriorate the healthy LC PUFA after they are ingested. Important lipid oxidation promoters in food are low molecular weight (LMW) iron (Fe) and methemoglobin (metHb). To incorporate the LC omega-3 PUFA safely into food with respect to oxidation, it is necessary to understand both Fe- and metHb-mediated oxidation of PUFA and how the oxidation is influenced by conditions and dietary antioxidants.The main objective of this thesis is therefore to study Fe- and metHb-mediated lipid oxidation in food-related lipid model systems – emulsions stabilised with phospholipids and liposomes made of phospholipids – containing LC omega-3 PUFA from fish. The focus was on clarifying the reaction mechanisms and the impact of different factors, including dietary antioxidants and gastric juice, on the prooxidant activity of Fe and metHb. Measurement of the consumption rate of the essential substrate for lipid oxidation – oxygen – by the LC PUFA was used for assessment of lipid oxidation.The continuous measurement of the dissolved oxygen concentration has been shown to be a robust method for direct and instantaneous monitoring of peroxidation in both the liposomes and emulsions. The method was especially useful for measurement of the oxygen consumption kinetics in the lipid systems. The determination of oxygen uptake rates (OUR) enabled screening and evaluation of the impact of the different factors and antioxidants on the prooxidant activity of Fe and metHb.Pre-formed lipid hydroperoxides (LOOH) were shown to be essential for the prooxidant activity of both Fe and metHb, and the prooxidant activity of metHb was not affected by the lack of light. The oxygen uptake kinetics revealed that iron behaved as a catalyst in lipid oxidation while the prooxidant activity of metHb weakened over time, presumably due to degradation of meHb molecule during lipid oxidation. MetHb was shown to be a stronger prooxidant than Fe, but the strong prooxidative activity was facilitated by a complete structure of the metHb molecule. The prooxidant mechanism of both Fe and metHb was not limited by the level of dissolved oxygen, as long as oxygen was present, or the level of pre-formed LOOH and double bonds in fatty acids, as long as they were present in higher concentrations than the prooxidant.The extent of the prooxidative activity of Fe was shown to vary in dependence on:The total surface area: Smaller liposomal vesicles with lower lipid content were more prone to oxidation than larger emulsion droplets with a higher lipid content, presumably due to more frequent interactions of Fe with pre-formed LOOH in the interphase.The amount of phospholipid emulsifier: Higher levels of phospholipids resulted in the formation of smaller droplets. The highest OUR were measured for emulsifier concentrations ranging from 5 – 10% (w/w lipid base).pH of the aqueous phase: Fe-mediated oxidation was highest at pH interval 4.5 – 5.5.Dissolved compounds: Sodium chloride (NaCl) and 0.2% of xanthan gum dissolved in the aqueous phase inhibited Fe-mediated oxidation in a concentration dependent manner.Electrostatic retention of Fe by phosphate groups within phospholipid heads has been suggested to facilitate the contact between pre-formed LOOH and Fe, and to create competitive reactions for iron precipitation at pH > 5 and iron complexation by chelating compounds.The activity of dietary antioxidants has been shown to be affected by the type of prooxidant in the lipid system. Ascorbic acid, caffeic acid, propyl gallate, astaxanthin, ascorbyl palmitate, α-tocopherol, and δ-tocopherol inhibited metHb-mediated oxidation in concentration dependent manners. EDTA had a minor effect on metHb-mediated oxidation.In Fe-mediated oxidation, caffeic acid, ascorbic acid and α-tocopherol were prooxidants. They directly interacted with Fe, reducing Fe3+ to the more catalytically active Fe2+. The magnitude of the pro-oxidative behaviour was dependent on the Fe-to-antioxidant ratio, antioxidant concentration and pH. Ascorbic acid was depleted by interactions with Fe, and decreased the pro-oxidative activity of α-tocopherol. EDTA and citric acid inhibited Fe-mediated oxidation completely at twice the ratio to Fe and pH > 3.5. Propyl gallate efficiently inhibited Fe-mediated oxidation, while astaxanthin and β-carotene had only minor effects. In addition, chemical structure and physical location of the antioxidants determined their effects.The work in this thesis shows that for correct interpretation of the effects of antioxidants it is important to assess what types of prooxidants are present in the system.Both gastric juice and hydrochloric acid solution (HCl) did not prevent oxidation of marine lipids in emulsions and liposomes (pH 4.0). Furthermore, gastric juice did not inhibit metHb-mediated oxidation, but it was capable of reducing the prooxidant activity of dietary LMW iron, compared to HCl solution. Berry juice, green tea, red wine, and caffeic acid reduced the OUR in the acidic environments while coffee, ascorbic acid and orange juice increased the OUR. Therefore, beverages accompanying foods rich in marine lipids will affect the course of post-prandial lipid oxidation.

Simple Technologies for Converting Rest Raw Materials of Atlantic Salmon (Salmo salar) into High-Quality, Valuable, and Tasty Feed Ingredients

ABSTRACT Rest raw materials as viscera, heads, and frames from farmed Atlantic salmon (Salmo salar) were hydrolyzed with the use of endogenous enzymes and the commercial enzymes Protamex and a mixture of Papain and Bromelain. Composition of the prehydrolysis mixture clearly influenced the quality of final hydrolysate and process kinetics. An increased proportion of viscera increased the amount of endogenous enzymes, which influenced hydrolysis kinetics, the extent of hydrolysis, and increased bitterness. Commercial enzymes, in addition to endogenous enzymes, are not always more yield efficient or economically beneficial but can be used to improve the taste and to ease the separation of oil from the hydrolysate fraction. Hydrolysis of denatured proteins in rest raw materials was hardly detectable. Optimum temperature should therefore be selected to avoid protein denaturation and simultaneously maintain high enzymatic activity. Hydrolysates preferably contain a low concentration of oil, and this work shows that high-quality oil can be separated before hydrolysis by mild thermal treatment. The previous separation of oil did not influence hydrolysate yield and decreased the concentration of lipids in the final hydrolysate. The initial separation of oil also increased productivity of the hydrolysis reactor, due to the reduction of hydrolysis volume.

Effect of dietary antioxidants on iron-mediated peroxidation in emulsions studied by dissolved oxygen consumption

Long chain omega-3 polyunsaturated fatty acids (LC omega-3 PUFA) are vital for physiological functions and have therapeutic and health benefits. The consumption of LC PUFA in the Western world has been below recommended intake levels the past decades, despite promotion of seafood and omega-3 supplements. Incorporation of the LC PUFA into processed food consumed on a daily basis might therefore bridge the gap between the recommended and actual consumption of LC omega-3 PUFA. Unfortunately, the development of omega-3 enriched food is hampered by a very high susceptibility of LC PUFA to oxidative deterioration. Furthermore, oxidised lipids are believed to create health risks. It has also been suggested that gastric juice may deteriorate the healthy LC PUFA after they are ingested. Important lipid oxidation promoters in food are low molecular weight (LMW) iron (Fe) and methemoglobin (metHb). To incorporate the LC omega-3 PUFA safely into food with respect to oxidation, it is necessary to understand both Fe- and metHb-mediated oxidation of PUFA and how the oxidation is influenced by conditions and dietary antioxidants.The main objective of this thesis is therefore to study Fe- and metHb-mediated lipid oxidation in food-related lipid model systems – emulsions stabilised with phospholipids and liposomes made of phospholipids – containing LC omega-3 PUFA from fish. The focus was on clarifying the reaction mechanisms and the impact of different factors, including dietary antioxidants and gastric juice, on the prooxidant activity of Fe and metHb. Measurement of the consumption rate of the essential substrate for lipid oxidation – oxygen – by the LC PUFA was used for assessment of lipid oxidation.The continuous measurement of the dissolved oxygen concentration has been shown to be a robust method for direct and instantaneous monitoring of peroxidation in both the liposomes and emulsions. The method was especially useful for measurement of the oxygen consumption kinetics in the lipid systems. The determination of oxygen uptake rates (OUR) enabled screening and evaluation of the impact of the different factors and antioxidants on the prooxidant activity of Fe and metHb.Pre-formed lipid hydroperoxides (LOOH) were shown to be essential for the prooxidant activity of both Fe and metHb, and the prooxidant activity of metHb was not affected by the lack of light. The oxygen uptake kinetics revealed that iron behaved as a catalyst in lipid oxidation while the prooxidant activity of metHb weakened over time, presumably due to degradation of meHb molecule during lipid oxidation. MetHb was shown to be a stronger prooxidant than Fe, but the strong prooxidative activity was facilitated by a complete structure of the metHb molecule. The prooxidant mechanism of both Fe and metHb was not limited by the level of dissolved oxygen, as long as oxygen was present, or the level of pre-formed LOOH and double bonds in fatty acids, as long as they were present in higher concentrations than the prooxidant.The extent of the prooxidative activity of Fe was shown to vary in dependence on:The total surface area: Smaller liposomal vesicles with lower lipid content were more prone to oxidation than larger emulsion droplets with a higher lipid content, presumably due to more frequent interactions of Fe with pre-formed LOOH in the interphase.The amount of phospholipid emulsifier: Higher levels of phospholipids resulted in the formation of smaller droplets. The highest OUR were measured for emulsifier concentrations ranging from 5 – 10% (w/w lipid base).pH of the aqueous phase: Fe-mediated oxidation was highest at pH interval 4.5 – 5.5.Dissolved compounds: Sodium chloride (NaCl) and 0.2% of xanthan gum dissolved in the aqueous phase inhibited Fe-mediated oxidation in a concentration dependent manner.Electrostatic retention of Fe by phosphate groups within phospholipid heads has been suggested to facilitate the contact between pre-formed LOOH and Fe, and to create competitive reactions for iron precipitation at pH > 5 and iron complexation by chelating compounds.The activity of dietary antioxidants has been shown to be affected by the type of prooxidant in the lipid system. Ascorbic acid, caffeic acid, propyl gallate, astaxanthin, ascorbyl palmitate, α-tocopherol, and δ-tocopherol inhibited metHb-mediated oxidation in concentration dependent manners. EDTA had a minor effect on metHb-mediated oxidation.In Fe-mediated oxidation, caffeic acid, ascorbic acid and α-tocopherol were prooxidants. They directly interacted with Fe, reducing Fe3+ to the more catalytically active Fe2+. The magnitude of the pro-oxidative behaviour was dependent on the Fe-to-antioxidant ratio, antioxidant concentration and pH. Ascorbic acid was depleted by interactions with Fe, and decreased the pro-oxidative activity of α-tocopherol. EDTA and citric acid inhibited Fe-mediated oxidation completely at twice the ratio to Fe and pH > 3.5. Propyl gallate efficiently inhibited Fe-mediated oxidation, while astaxanthin and β-carotene had only minor effects. In addition, chemical structure and physical location of the antioxidants determined their effects.The work in this thesis shows that for correct interpretation of the effects of antioxidants it is important to assess what types of prooxidants are present in the system.Both gastric juice and hydrochloric acid solution (HCl) did not prevent oxidation of marine lipids in emulsions and liposomes (pH 4.0). Furthermore, gastric juice did not inhibit metHb-mediated oxidation, but it was capable of reducing the prooxidant activity of dietary LMW iron, compared to HCl solution. Berry juice, green tea, red wine, and caffeic acid reduced the OUR in the acidic environments while coffee, ascorbic acid and orange juice increased the OUR. Therefore, beverages accompanying foods rich in marine lipids will affect the course of post-prandial lipid oxidation.