Progress 08/01/16 to 03/31/18
Outputs
Target Audience:The target audiences reached at the beginning of this phase I project included renderers as members of the Natonal Renderers Association and as members of the Fats and Protein Research Foundation both of which hosted discussions with us about our projects. Also, individual renderers were visited during which this project and potential product was discussed. In addition, several companies with interests in food grade antioxidants were approached and discussions centered upon our potential product and its advantages. These companies included a potential distributor of our product. Also, other researchers with a history of work on antioxidants were consulted about this project. An equipment manufacturing company has helped us with suggestions for various steps in the manufacturing stream that will provide for more efficient yields. Lastly contact with a leading manufacturer of pre-cooked products for which this antioxidant is intended led to the building of a future relationship. Changes/Problems:With our antioxidant in the efficacy studies, we observed poorbatch-to batch reproducibilityof the efficacy, with somebatches working well andperforming on par withcommercial antioxidants, andother batches not showing anyconsiderable protection. Lack of reproducibility was noticed even when higherapplication rates of 0.5% wt.were used. We have previously evaluatedour antioxidant in a number ofraw meat models as well asother food and feed models(liquid oils, protein meals, fat-coated kibble, etc.), and never observed such a lack ofreproducibility. We noticed one difference between the previously used models and currently usedcooked meat models: a lot of water is excreted from ground beef and chicken during cooking, andthis water is lost when a cooked sample is collected for storage. In fact, average loss of weightduring cooking was 30±2.5% due to discarded excess liquid and evaporated water. Wehypothesized that our water-soluble antioxidant cansometimes be lost with this excreted water, leading to at leastpartial and sometimes complete loss of efficacy. In order toreduce the loss of our antioxidant with excreted water, wedecided to make a fat-soluble formulation of our product. Toachieve that we decided to use lecithin,a well-knownemulsifier, which is broadly used in food industry. Since purelecithin did not readily form a homogeneous mixture with ourantioxidant (perhaps due to high concentration of zincchloride in the latter), we also added vegetable oil to improvehomogenization.The resulting homogeneous product is a viscous opaque liquid that has consistency of a thick honey and light pleasant odor.We noticed that upon application to ground meat, our fat-soluble formulation is quickly absorbedby the meat, presumably spreading into the fat phase. Efficacy studies showed that this fat-soluble formulation was consistently efficient in cooked beef and chicken models. Its efficacy wason par with that of the commercial antioxidants. Thus, adding lecithin helped to increasemiscibility of our antioxidant with fat and prevent its loss with excreted water, leading to improved reproducibility. What opportunities for training and professional development has the project provided?
Nothing Reported
How have the results been disseminated to communities of interest?One of the primary target audiences of this work is the rendering community whether it is independent renderers or packer renderers. We have presented our findings routinely and periodically to their Fats and Protein Research Foundation.as well as at their annual conference. In addition, several members have been contacted personally, visits arranged, and presentations made on the results obtained. Also, results have been shared with distributors, potential investors, equipment manufacturing companies, etc. What do you plan to do during the next reporting period to accomplish the goals?
Nothing Reported
Impacts
What was accomplished under these goals?
Objective I: Detailed efficacy study. The goal of this Phase I study was to evaluate our antioxidant in cooked meat modelsalso measuring organoleptic properties important for consumption, such as color, odor, and texture in several human food models. To better understand these effects,a series of experiments with ground beef and ground chicken were conducted.Ground beef was selected as a meat source for these experiments as lipid oxidationoccurs more rapidly in a ground meat product where the muscle fiber cells may be lysed duringgrinding to expose the phospholipid component of the muscle fiber lipid bilayer. This has beenshown to be a key component in initiating lipid oxidation. Ground chicken was selected as chicken meat is more susceptible to lipid oxidation due to higher levels of polyunsaturated fatty acids whichoxidize more rapidly. Additionally, chicken hasa milder flavor profile than beef. If flavor differences were induced by the addition of our erythrocyte protein extract, flavor due to our antioxidant would be more apparent. Thechanges in TBARS values indicate that for both precooked ground beef and chicken, the meat system used induced high levels of lipidoxidation. Use of commercial antioxidants, rosemary and BHA/BHT, kept the TBARS levels below 2.5 at day 3, indicative of good antioxidantefficacy. With our antioxidant, however, we observed poor batch-to batch reproducibility, with some batches working well andperforming on par with commercial antioxidants, and other batches not showing any considerable protection. Lack of reproducibility was noticed even when higher application rates of 0.5% wt.were used. We have previously evaluated our antioxidant in a number of raw meat models as well as other food and feed models (liquid oils, protein meals, fat-coated kibble, etc.), and never observed such a lack of reproducibility. We noticed one difference between the previously used models and currently used cooked meat models: A lot of water is lost from ground beef and chicken during cookingand this water is lost when a cooked sample is collected for storage. Wehypothesized that our water-soluble antioxidant can sometimes be lost with this excreted water, leading to at least partial and sometimes complete loss of efficacy. In order to reduce the loss of our antioxidant with excreted water, we decided to make a fat-soluble formulation of our product. Toachieve that we decided to use lecithin - a well-known emulsifier, which is broadly used in food industry. Since pure lecithin did not readily form a homogeneous mixture with our antioxidant (perhaps due to high concentration of zincchloride in the latter), we also added vegetable oil to improve homogenization. The resulting homogeneous product is a viscous opaque liquid that has consistency of a thick honey and light pleasant odor. We noticed that upon application to ground meat, our fat-soluble formulation is quickly absorbed by the meat, presumably spreading into the fat phase. Efficacy studies showed that this fat-soluble formulation was consistently efficient in cooked beef and chicken models. Its efficacy was on par with that of the commercial antioxidants. Thus, adding lecithin helped to increase miscibility of our antioxidant with fat and prevent its loss with excreted water, leading to improved reproducibility. There were no meaningfuldifferences by storage day for all other treatment groups, either in pre-cooked beef or chickenmodel. These results indicate that the addition of the erythrocyte protein extract, either with or without lecithin, did not appreciably affect the aroma of pre-cooked beef and chicken crumbles and can therefore be applied to multiple meat products without impacting flavor. Conclusions for Objective 1. We developed a fat-soluble formulation of our antioxidant, which wasconsistently efficient in cooked beef and chicken meat models at the application rate of 0.25% wt.without affecting the aroma of the meat. Objective II: Mechanism of Action. Our initial hypothesis was that antioxidant activity of our product is mostly due to enzymaticsuperoxide dismutase and/or catalase activity. These two enzymes are abundant in red blood cells and are known potent scavengers of reactive oxygen species. These two enzymes are not the major factors contributing to antioxidant activity of our product, although they may work synergistically with other antioxidants present in red blood cells. Another potent antioxidant present at relatively high levels in red blood cells in glutathione. Our product at only 0.18% application rate shows higher efficacy against oxidation than pure glutathione at 1 g/L. Of note, the physiological concentration of glutathione in red blood cells ranges between about 0.3 g/L and 1 g/L. Therefore, the glutathione concentration in cod oil treated by 0.18% of our product should not exceed 0.0018 g/L. As such, our product provides more than 500-fold better protection than pure glutathione in preserving a food product. Consequently, we hypothesized that antioxidant activity of our product is due to synergistic actionof several antioxidants. In fact, in living cells reduced form of glutathione is constantly replenished by a number of enzymes, most important of which is glutathione reductase. Other enzymes involved in glutathione cycle are also potent antioxidants (e.g., glutathione peroxidase). To better understand which antioxidant makes most significant contribution to the overall efficacy of our product, we separated it into low- and high- molecular weight fractions.. Efficacy of low- and high-molecular weight fractions was then compared to that of the same batch of the whole product in a cod oil model. The low molecular weight fraction shows much higher antioxidant activity than the high molecular weight fraction, although even low molecular weight fraction was not as efficient as the wholeproduct. These results indicate that glutathioneis likely the major active component of our product, while other components are probably involved in glutathionerecycling (e.g., glutathione reductase) and provide synergy by scavenging on reactive oxygenspecies, or by using glutathione as a substrate to breakdown reactive oxygen species. Another lowmolecular weight antioxidant present in erythrocytes is vitamin E, but it is poorly water soluble and present predominantly in cell membrane, so it is unlikely to be present in the low molecular weight fraction. Conclusions for Objective 2. Our results indicate that glutathione is likely the major active component of our product, while higher molecular weight components may provide synergy by recycling glutathione and breaking down additional reactive oxygen species. Objective III: Evaluation of susceptibility to microbiological contamination. Possible contamination with pathogens presents an important safety concern. In a preliminarystudy we noticed that our antioxidant was resistant to challenges by a model pathogen,Staphylococcus aureus, possibly due to high concentration of zinc chloride (15-20 wt. %) present in the system. The goal of this Objective was to expand this study to include challenges by other common pathogens that could be present in animal or poultry products. We also measured water activity for three independent batches of aqueous formulation of our antioxidant. Values of water activity were between 0.79 and 0.84 for all batches. In general, most bacteria cannot grow below a water activity of 0.90 to 0.91 except for some of the most halotolerant species and aerobic Staphylococcus aureus (for the latter, minimum water activity level is 0.86). Conclusions for Objective 3. Our new fat-soluble formulation showed remarkable resistance tomicrobiological challenge and efficiently inhibited growth of three common pathogens: C. jejuni, L. monocytogenes, and S. enterica. Results of water activity experiments indicate that our product is unlikely to be supportive of growth of other pathogens.
Publications
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Progress 08/01/16 to 07/31/17
Outputs
Target Audience:The target audiences reached at the beginning of this phase I project included renderers as members of the Natonal Renderers Association and as members of the Fats and Protein Research Foundation both of which hosted discussions with us about our projects. Also, individual renderers were visited during which this project and potential product was discussed. In addition, several companies with interests in foodgrade antioxidants were approached and discussions centered upon our potential product and its advantages. These companies included a potential distributor of our product. Lastly, other researchers with a history of work on antioxidants were consulted about this project. Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided?
Nothing Reported
How have the results been disseminated to communities of interest?Presentations were made to the Fats and Protein Research Foundation and to the National Renderers Association at their annual meetings. What do you plan to do during the next reporting period to accomplish the goals?We will continue wih the work toward the goals in Aims 2 and 3. Specifically, we willcompare antioxidant activity of our product to that of known individual antioxidant components present in blood. According to the literature, glutathione and vitamin E are two most potent antioxidants that prevent lipid oxidation in erythrocytes in vivo. We therefore will focus on these two molecules as the most likely candidates responsible for antioxidant activity in food models. Also, for Aim 3, we are concerned about possible contamination with pathogens which presents an important safety concern. However, in a preliminary study we noticed that CP was resistant to challenges by a model microorganism,Staphylococcus aureus, possibly due to high concentration of zinc chloride (15-20 wt. %) present in the system. Here, we propose to expand this study to include challenges by the most common pathogens that could be present in animal or poultry products,E.ColiandSalmonella enteridis.
Impacts
What was accomplished under these goals?
Results for Aim 1: The results indicate that the Clemson antioxidant was providing some lipid oxidation protection and had minimal effect on the color of raw and cooked beef crumbles. The results show that the system provided sufficient opportunity for lipid oxidation development. TBARS values greater than 2.0 to 2.5 in cooked beef are generally defined as oxidized. The addition of rosemary and BHA/BHT, commonly used antioxidant in beef, chicken and pork processed products, resulted in low TBARS values, below 1.0, throughout storage indicating that these commonly used antioxidants retarded lipid oxidation. The results indicated that the Clemson antioxidant help to provide protection on the development of lipid oxidation in pre-cooked beef crumbles when added at higher level, 0.50%, for up to 3 days. These results do not provide understanding of why the Clemson antioxidant did not control as effectively as the use of rosemary or BHA/BHT. However, lipid oxidation was retarded with the use of the Clemson antioxidant. Pre-cooked chicken crumbles containing the low level of Clemson antioxidant has slightly lower TBARS values than control pre-cooked chicken crumbles indicating that the use of the low level of Clemson antioxidant did not provide sufficient antioxidant protection. However, when the Clemson antioxidant was used at a higher level, lipid oxidation was retarded. The results indicate that the addition of the Clemson antioxidant at levels used in this study did not appreciably affect the aroma of pre-cooked beef and chicken crumbles stored up to 5 days in an aerobic environment at 4°C. Conclusions for Aim 1. The addition of Clemson antioxidant to ground beef and chicken had minimal effects on raw and cooked pH and color attributes. Additionally, the use of the Clemson antioxidant did not appreciably impact aroma of pre-cooked beef and chicken crumbles. However, the Clemson antioxidant used at the higher level, 0.50%, had antioxidant properties. While these properties were not as affective as the use of rosemary and BHA/BHT, current commonly used meat antioxidants, these results are promising. The use of the Clemson antioxidant at either higher levels or formulation of the antioxidant to maintain functionality with subsequent storage would be recommended. Results for Aims 2 and 3: Work is still in progress fro Aims 2 and 3. We request a no cost extension for this project for one year to complete the studies proposed in these Aims.
Publications
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Progress 08/01/16 to 03/31/17
Outputs
Target Audience:The target audiences reached at the beginning of this phase I project included renderers as members of the Natonal Renderers Association and as members of the Fats and Protein Research Foundation both of which hosted discussions with us about our projects. Also, individual renderers were visited during which this project and potential product was discussed. In addition, several companies with interests in foodgrade antioxidants were approached and discussions centered upon our potential product and its advantages. These companies included a potential distributor of our product. Lastly, other researchers with a history of work on antioxidants were consulted about this project. Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided?
Nothing Reported
How have the results been disseminated to communities of interest?Presentations were made to the Fats and Protein Research Foundation and to the National Renderers Association at their annual meetings. What do you plan to do during the next reporting period to accomplish the goals?We will continue wih the work toward the goals in Aims 2 and 3. Specifically, we willcompare antioxidant activity of our product to that of known individual antioxidant components present in blood. According to the literature, glutathione and vitamin E are two most potent antioxidants that prevent lipid oxidation in erythrocytes in vivo. We therefore will focus on these two molecules as the most likely candidates responsible for antioxidant activity in food models. Also, for Aim 3, we are concerned about possible contamination with pathogens which presents an important safety concern. However, in a preliminary study we noticed that CP was resistant to challenges by a model microorganism,Staphylococcus aureus, possibly due to high concentration of zinc chloride (15-20 wt. %) present in the system. Here, we propose to expand this study to include challenges by the most common pathogens that could be present in animal or poultry products,E.ColiandSalmonella enteridis.
Impacts
What was accomplished under these goals?
Results for Aim 1: The results indicate that the Clemson antioxidant was providing some lipid oxidation protection and had minimal effect on the color of raw and cooked beef crumbles. The results show that the system provided sufficient opportunity for lipid oxidation development. TBARS values greater than 2.0 to 2.5 in cooked beef are generally defined as oxidized. The addition of rosemary and BHA/BHT, commonly used antioxidant in beef, chicken and pork processed products, resulted in low TBARS values, below 1.0, throughout storage indicating that these commonly used antioxidants retarded lipid oxidation. The results indicated that the Clemson antioxidant help to provide protection on the development of lipid oxidation in pre-cooked beef crumbles when added at higher level, 0.50%, for up to 3 days. These results do not provide understanding of why the Clemson antioxidant did not control as effectively as the use of rosemary or BHA/BHT. However, lipid oxidation was retarded with the use of the Clemson antioxidant. Pre-cooked chicken crumbles containing the low level of Clemson antioxidant has slightly lower TBARS values than control pre-cooked chicken crumbles indicating that the use of the low level of Clemson antioxidant did not provide sufficient antioxidant protection. However, when the Clemson antioxidant was used at a higher level, lipid oxidation was retarded. The results indicate that the addition of the Clemson antioxidant at levels used in this study did not appreciably affect the aroma of pre-cooked beef and chicken crumbles stored up to 5 days in an aerobic environment at 4°C. Conclusions for Aim 1. The addition of Clemson antioxidant to ground beef and chicken had minimal effects on raw and cooked pH and color attributes. Additionally, the use of the Clemson antioxidant did not appreciably impact aroma of pre-cooked beef and chicken crumbles. However, the Clemson antioxidant used at the higher level, 0.50%, had antioxidant properties. While these properties were not as affective as the use of rosemary and BHA/BHT, current commonly used meat antioxidants, these results are promising. The use of the Clemson antioxidant at either higher levels or formulation of the antioxidant to maintain functionality with subsequent storage would be recommended. Results for Aims 2 and 3: Work is still in progress fro Aims 2 and 3. We request a no cost extension for this project for one year to complete the studies proposed in these Aims.
Publications
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