NZ720227B2 - Antioxidant formulations - Google Patents

Abstract

Antioxidant formulations containing new active molecules with tocopherols are disclosed. The best performing formulas contain extracts of green tea that are oil soluble, extracts of rosemary, extracts of spearmint and tocotrienols. Interestingly, the amount of tocopherols in formulas could be reduced by 50% in this diet when the other actives were increased accordingly. d by 50% in this diet when the other actives were increased accordingly.

Description

ANTIOXIDANT FORMULATIONS Background of the ion This application claims priority to United States Patent ation Serial No. 61/532,859, filed September 9, 2011, which is incorporated herein in its entirety by this reference. This application is a divisional application of New Zealand Patent application No 65958, which is incorporated herein in its entirety by reference.

The present invention relates generally to antioxidant formulations containing extracts of tea and extracts of spearmint and, more specifically, to antioxidant formulations for pet food containing lipid-soluble extracts of tea and water-soluble extracts of a Lamiaceae spp. plant such as spearmint containing, inter alia, rosmarinic acid.

Antioxidants are applied at several stages of the pet food kibble manufacturing process , , and after extrusion. One common antioxidant ses mixed tocopherols and/or tocotrienols. In one particular example, Naturox® Plus Dry (Kemin Industries, Inc., Des Moines, Iowa) is a dry idant (DA) mixture which is added to the kibble dry recipe before it is extruded, while Naturox® Premium Liquid (Kemin ries, Inc., Des Moines, Iowa) is a liquid antioxidant (LA) formulation in oil added to the enrobing fat on the 's surface.

Since LA is d closest to the air/kibble interface, it is crucial to the oxidative stability of the kibble. The LA formulation may also be applied to the meat meal during its production in the rendering process or directly after the rendering when the meal is isolated from the offal, to control oxidation of protein and fat prior to be used in the dry meal.

Green and black teas, as well as other varieties of tea, are well known to have water soluble antioxidants which perform well in a hydrophilic food matrix. Previous attempts at suspending these water soluble antioxidants in oil with AAFCO (Association of American Feed Control Officials) approved ingredients at concentrations needed for use in antioxidant formulations have been unsuccessful.

Summary of the Invention Recently, lipid soluble catechins (LSC) were identified to have antioxidant properties and maintain solubility in hydrophobic media, including ble oils. Oxidative Stability Index (OSI) results of LSC in animal and vegetable fat were promising and, as a , the material was used in this sequence of trials. These materials, in conjunction with rosemary extract, AH26(11310462_1):JIN natural surfactants (lecithin) and ors c acid), were paired in formulas with the goal of creating a more versatile and equal, if not improved, formula with better efficacy.

New dry antioxidant formulations were developed to include not only fat soluble antioxidants, chelators, tants, but water e antioxidant extracts from spearmint and rosemary, which provided for an improved level of efficacy, especially at rated temperature storage conditions. As part of the idant dry formulations, the chelators were varied and included c acids, nic phosphate, milk whey protein and polyphosphate chelators with a ed range in pH from 1.5 to 12. Surfactants for the dry formulations included lecithin, but also may include other ionic or non-ionic surfactants that are naturally derived or from non-natural sources, to either solubilize, act as a synergist and enhancing the antioxidant properties, or to further distribute the antioxidant into the desired host matrix.

For all formulations mixed tocopherols includes a single iso-form or a mixture of all structural s (alpha, beta, gamma, etc.) of tocopherols and/or tocotrienols. [007A] Thus, in one aspect of the present invention there is provided animal feed or a constituent of animal feed comprising a tocopherol and a rosmarinic acid-containing extract of a Lamiaceae spp. plant.

Brief Description of the Drawings Fig. 1 is a chart of peroxide values (meq/kg diet) of kibbles treated with 1000 ppm of 8 different liquid antioxidant formulations stored at 65°C Fig. 2 is a comparison between OSI and PV (time to rancidity) results in sunflower oil treated with idants Description of the Invention The best antioxidant choice for stabilization of any given product depends on multiple complex factors. One of the factors that s the selection is the polarity of the antioxidant.

The polarity of the antioxidant affects where the antioxidant is located in the product and whether it can interact with free radicals. For example, polar antioxidants are effective in bulk oil situations due to what is referred to as the polar paradox. The polar paradox states that polar antioxidants do not like a lar oil matrix and will concentrate at polar interfaces. In a bulk oil these interfaces include air/oil interfaces and water/oil interfaces often in the form of emulsified micelles. A non-polar antioxidant is not as effective as the antioxidant is simply diluted and dispersed in the general oil matrix and not concentrated at the interface. The opposite has been observed with animal n meals and pet food diets, where lar AH26(13851987_1):MXC antioxidants have been observed to perform the best. Numerous trials have shown that the polar antioxidants do not perform as well in these meal and diet matrices.

Previous work has attempted to e the top antioxidants that performed well in the AOCS Official Method Cd 12b—92 oil stability index (081) to identify the best antioxidants for stabilization. As expected in an 081 test which measures stability on bulk oils, the polar antioxidants perform the best due to the polar paradox described earlier. It is common practice in the industry to use the 081 as an antioxidant screening tool. It has been our observations that the 081 is not an appropriate tool for predicting the best antioxidants for meals or diets, and goes r to accepted practice. Top performing polar antioxidants include examples such as water soluble green tea, gallic acid, ascorbic acid, etc. In particular, water soluble green tea extracts contain a substantial quantity of the unmodified or natural leaf polyphenols many of which are synthesized into the natural form of catechins (see, e.g., US 2007/0286932). These antioxidants perform very poorly when they are used for meal and diet stabilization. While the best antioxidants for meals and diets are lar, there are a limited number of natural non—polar or oil soluble antioxidants. Examples include tocopherols, tocotrienols, camosic acid, etc. There are other non—polar antioxidants but they do not have favorable commercial pricing. The present invention discloses for the first time the use of lipid soluble catechins which have the age of being oil soluble, economically viable and suitable for replacing a large amount of conventional antioxidants while still providing effective stabilization of meals and diets. The lipid soluble catechins are shown to e meal and diet stabilization beyond what is achievable in water soluble forms of catechins.

In the present invention, the antioxidants, individually or in combination, can be added to the l diet or to the oil used in the diet. erols are ionally applied to diets in s between 50 and 250 ppm.

Tocopherols are known to be prooxidants in oils above about 5000 ppm. The ranges of tocopherols applied to oils and diets in the present invention are between 10 ppm and about 250 ppm with a preferred range or between 40 ppm and 240 ppm.

In the present invention, ry extracts are used in the range of between 0 and 100 ppm to the diet, with a preferred range of between 0 ppm and 60 ppm to the diet, and between 0 ppm and 360 ppm to the oil with a preferred range of between 0 ppm and 200 ppm to the oil.

In the present invention, lipid soluble catechins are used in the range of between 0 and 120 ppm to the diet, with a preferred range of between 10 ppm and 60 ppm to the diet, and between 10 ppm and 150 ppm to the oil with a preferred range of between 10 ppm and 75 ppm to the oil.

Example 1 — Addition of Lipid Soluble Tea Extract Materials and Methods Liquid antioxidant formulas, the compositions of which are listed Table l, were d to extruded kibble in enrobing fat.

Table 1. Active ingredients of liquid antioxidant formulations.

Treatment Tocopherols Rosemary LSC Name (%) (%) (%) LA 1 0 0 0 LA 2 24 0.1 0 LA 3 20 0.1 2 LA 4 17 0.1 5 The chicken fat was treated with 3000 ppm of the liquid antioxidant formulas prior application to the kibble at 4.5%. Palatant was also applied to the kibble at 1%. ed kibble was stored at 47 °C in individual plastic bags and analyzed for peroxide values (PV) using the FOX II Method (Gulgun Yildiz, Randy L. Wehling and Susan L. Cuppett. Comparison of four analytical methods for the determination of de value in oxidized n oils. l of the American Oil ts' Society Volume 80, Number 2, 2003, 103—107; Nourooz—Zadeh, Jaffar; Tahaddine—Sarmadi, Javad; ez—Aragon, Ines; and Wolff, Simon P. Measurement of Hydroperoxides in Edible Oils Using the Ferrous Oxidation in Xylenol Orange Assay. J. Agric Food Chem, B01 43. No. l. 1995, 17—21) every 2 weeks. Formation of hexanal and 2,4— decadienal was measured at week 4 by gas chromatography (Frankel, EN. Methods to ine extent of oxidation. In: Lipid Oxidation. The Oily Press: Dundee, Scotland. Copyright 1998).

The results are presented in Table 2.

Table 2. de values and aldehydes (sum of hexanal and 2,4—decadienal) levels of the kibble stored at 47 °C for 4 weeks.

Treatment Name PV Aldehydes (mEq/kg sample) (ppm) LA 1 4.2 156 LA 2 2.1 69 LA 3 1.4 48 LA 4 1.3 43 After 4 weeks of storing the kibble at 47 °C the study was terminated as the peroxide values for all of the treatments reached 1 mEq/kg, which is ered an indication of rancidity.

As expected, lack of antioxidants (LAl) resulted in highest peroxide value as well as level of aldehydes. More importantly, formulas containing LSC outperformed tocopherol—based antioxidant, as apparent from the peroxides values (1.4, 1.3 for LA3, LA4 vs. 2.1 for LA2) and aldehydes content (48, 43 for LA3, LA4 vs. 69 for LA2).

Example 2 — Addition of Spearmint Extract Dry antioxidant formulations, listed in Table 3, were added to kibble dry mix with a ribbon r and extruded in sequence. Water soluble green tea extract (WSGT) standardized to 45% epigallocatechin gallate and 45% other catechins was obtained from Kemin Industries, Inc. (Des Moines, Iowa).

Table 3. Active ingredients of dry antioxidant ations.

Treatment Tocopherols Rosemary WSGT LSC Spearmint Name (%) (%) (%) (%) (%) DA 1 0 0 0 0 0 DA2 22 0.1 0 0 0 DA 3 11 5 6 0 0 DA4 11 5 0 6 0 DA 5 11 5 0 0 5 The s were coated with untreated chicken fat at 4.5% and palatant at 1%, and placed in storage at 25 0C, 37 °C and 47 OC. Samples were analyzed for peroxide values (PV) using the FOX 11 Method and secondary lipid oxidation products al and 2,4 decadienal) by gas chromatography. Results are shown in Table 4.

Table 4. Peroxide values and aldehydes (sum of hexanal and 2,4—decadienal) levels of the kibble stored at t temperature, 37 °C and 47 OC.

PV Aldehydes Treatment Name (mEq/kg sample) (ppm) Ambient (16 weeks) DA 1 1.9 57 DA 2 1.0 28 DA 3 1.6 44 DA 4 0.7 21 DA 5 0.7 18 37 0C (12 weeks) DA 1 7.0 296 DA 2 5.6 186 DA 3 5.8 234 DA 4 2.8 77 DA 5 1.5 38 47 °C (4 weeks) DA 1 4.2 156 DA 2 6.3 184 DA 3 5.3 187 DA 4 1.1 29 DA 5 1.1 26 The ion of dry antioxidant into the kibble results in higher oxidative stability as evident from lower peroxide values and aldehyde levels under the storage conditions.

Antioxidant as containing LSC and spearmint extract performed substantially better than the tocopherol—based formula, ally at higher temperatures. Interestingly, the LSC and WSGT containing ations demonstrated vast differences in performance, showing that the water—soluble green tea extract did not control oxidation in the pet food matrix tested.

Exam le 3 — Evaluation of Antioxidant Activit of Li id Soluble Tea Catechins LSC b 081 The effectiveness of LSC extract in combination with erols, rosemary extract, and lecithin was tested using an animal fat as a matrix. Formulations listed in Table 5 were applied to the fat at 1000 ppm and 3000 ppm levels.

Table 5. Composition of formulas Treatment Tocopherols Rosemary LSC Name (%) (%) (%) 0% LSC 22 0.1 0 1% LSC 21 0.1 1 2% LSC 20 0.1 2 3% LSC 19 0.1 3 % LSC 17 0.1 5 The ion period of the fat treated with antioxidant formulations (Table 6) was compared to the untreated fat.

Table 6. 081 results for chicken fat d with antioxidant formulas.

OSI (hr) Treatment Name 1000 ppm 3000 ppm Untreated 5.9 0% LSC 21.6 31.1 1% LSC 22.1 31.9 2% LSC 23.7 35.8 3% LSC 24.1 37.7 % LSC 25.4 42.9 081 results show that the antioxidant ty of the formulas increased with higher LSC content. Samples containing 5% LSC applied to the fat at 3000 ppm had the highest induction period among tested formulations.

Example 4 — Evaluation of Antioxidant Efficacy at High Temperatures Fat samples were treated with 1000 and 3000 ppm of mental antioxidant formulas haVing varying ratios of tocopherols, rosemary extract, lipid soluble tea catechins (LSC) and lecithin, as shown in Table 7, and tested in duplicate in the 081 at 100 9C (Table 8).

Table 7. LA Prototypes tested in the LSC storage study.

Treatment Name Tocopherols (%) Rosemary (%) LSC Lecithin (%) (%) LSC—1 0 0 0 0 LSC-2 24 0.1 0 0 LSC—3 12 6 3 2 LSC—4 18 0 4 2 LSC—5 15 0 7 2 LSC—6 12 0 10 2 LSC—7 0 12 12 2 LSC—8 0 5 18 2 Table 8. 081 results of LSC formulas in chicken fat.

OSI (h) Treatment 1000 ppm 3000 ppm Name LSC—1 6.7 6.7 LSC—2 30.7 56.7 LSC—3 35.7 62.5 LSC—4 35.7 54.0 LSC—5 39.1 57.1 LSC—6 16.4 34.4 LSC—7 14.0 30.6 LSC—8 34.6 50.7 Additionally, 9 g treated poultry fat was weighed into an 081 tube, stored in an 081 unit at 65 °C and connected to air flow . The progress of oxidation was measured by analyzing the rise in peroxide values over time e 1).

The performance of the liquid formulations containing LSC was equivalent or improved when tested in the 081 at 65 OC. Formula LSC—3 out—performed all other formulas at 65 OC, and was statistically equivalent in the 081 to the current Naturox® Premium Liquid.

Example 5 — Swergy Between Antioxidants Experiments were ted to study the effect of combination of antioxidants on the time to rancidity of sunflower oil. Sunflower oil was treated with tocopherol at 1200 ppm alone and combined with WSGT (350 ppm), ry extract (250 ppm) and LSC (350 ppm) and placed in an incubator at 40 9C. Samples of the sunflower oil were ically analyzed for de values (PV) using the FOX 11 Method. Time to rancidity (PV210 meq/kg oil) was determined for all of the treatments. Results show the increase in stability of sunflower oil treated with combinations of antioxidants (Table 9) in contrast to the treatment with tocopherols alone.

Table 9. Synergistic Effect of Antioxidant Combinations on Time to Rancidity Time to rancidity Stability (days) se Tocopherol (1200 ppm) 9 Tocopherol (1200 ppm) + WSGT (350 ppm) 14 56% Tocopherol (1200 ppm) + Rosemary (250 ppm) 21 133% Tocopherol (1200 ppm) + LSC (350 ppm) 28 211% Tocopherols are known to be especially effective in stabilizing sunflower oil. However, a marked and unexpected increase in stability was observed with the addition of lipid soluble catechins. This increase is significantly longer than what was observed with the water soluble green tea (WSGT) and is counter to what was observed by the OSI results. e 6 — Comparison of ity of Sunflower Oil in OS1 and PV Score According to the American Oil Chemist Society, the Oil Stability Index (OSI) is the point of maximum change in an oil of fat’s oxidation under standard conditions. Accordingly, the OSI determines the relative resistance of an oil or fat to oxidation and is an indicator of the length of shelf life for that fat or oil. ments were done to evaluate the effect of lipid soluble catechins on the OSI of sunflower oil and on the shelf life of sunflower oil. er oil was treated with four different antioxidants: tocopherol at 1200 ppm (total tocopherol concentration); rosemary at 250 ppm (RosanTM SF 35 from Kemin Industries, Inc., a rosemary extract standardized to 10% carnosic acid); water soluble green tea extract at 35 ppm (standardized to 45% EGCG and 45% other catechins); lipid soluble catechins at 35 ppm (standardized to 74% ins). Untreated sunflower oil was used at the control. A shelf life study of the same samples at ambient temperature was also conducted. Shelf life time to rancidity was d as the number of days before the peroxide value (PV) exceeded 10 meq/kg.

The results are set out in Table 10.

Table 10. Time to Rancidity of Sunflower Oil OSI Tlme to ranc1d1ty Name (h) (dayS) Untreated 11.45 9 Tocopherol (1200 ppm) 14.95 9 ry (250 ppm) 26.35 21 WSGT (350 ppm) 31.65 14 LSC (350 ppm) 19.15 35 The s show that, surprisingly, the 081 results were not predictive of shelf life for lipid soluble catechins (Fig. 2). The lipid soluble ins are much more effective at extending shelf life than was expected from the 081 results. e 7 — Synergy Between Antioxidants Experiments were conducted to study the effect of antioxidants alone and in combinations on the peroxide value and 2,4—decadienal values of kibble after 6 weeks at 37 0C.

In a first set of experiments, the poultry fat used to coat the kibble was either left untreated or treated with 240 ppm tocopherol, 50 ppm rosemary, 70 ppm WSGT, or 70 ppm LSC. The results are shown in Table 11. In a second set of experiments, the fat used to coat the kibble was either left untreated or treated with 240 ppm tocopherol plus 50 ppm rosemary extract, 240 ppm tocopherol plus 70 ppm WSGT, 240 ppm tocopherol plus 70 ppm LSC, 50 ppm rosemary plus 70 ppm WSGT, and 50 ppm rosemary plus 70 ppm LSC. The results are shown in Table 12.

Table 11 — Effect of Antioxidant Combinations on Peroxide and 2,4—Decadienal Values PV 2,4-Decadienal Treatment Name (mEq/kg sample) (mEq/kg sample) Untreated fat 19.2 22 240 ppm tocopherol 14.7 18 50 ppm rosemary 17.7 20 70 ppm WSGT 16.7 18 70 ppm WSGT base 17.7 20 70 ppm LSC 21.6 24 Table 12 — Effect of Antioxidant Combinations on de and 2,4—Decadienal Values PV 2,4-Decadienal Treatment Name (mEq/kg sample) (mEq/kg sample) Untreated fat 19.2 22 240 ppm tocopherol + 50 ppm rosemary 14.7 17 240 ppm tocopherol + 70 ppm WSGT 14.9 17 240 ppm tocopherol + 70 ppm WSGT base 15.7 19 240 ppm tocopherol + 70 ppm LSC 15.0 17 50 ppm tocopherol + 50 ppm WSGT 20.8 24 50 ppm tocopherol + 50 ppm WSGT base 17.1 19 50 ppm tocopherol + 70 ppm LSC 16.0 18 From Table 11 it is seen that the lipid soluble catechins when used alone did not perform as well as the other antioxidants and indeed did not m as well as leaving the fat untreated.

WSGT was one of the better performing antioxidants, which again matched with the observations from the OSI testing. However, when used in combination with tocopherol (Tables 9 and 12), the lipid e catechins provided a synergistic protective effect, enabling a reduction in erol to imately one—fifth of the prior inclusion level without icantly increasing either the peroxide or cadienal values. A key goal of the pet food industry has been to reduce the use of tocopherols in the formulations. It has previously been difficult to reduce tocopherol concentrations due to ulty finding synergistic antioxidants that are effective in a combination that matches the stabilization capability of tocopherols on products under real world storage conditions. In this work we’ve been able to reduce tocopherol levels up to 80% and still e similar or better shelf life on a finished pet food diet. This work has shown synergism between tocopherols and LSC in combination or in addition to other antioxidants.

The foregoing description and drawings comprise illustrative embodiments of the present inventions. The ing embodiments and the methods described herein may vary based on the ability, experience, and preference of those skilled in the art. Merely listing the steps of the method in a certain order does not constitute any limitation on the order of the steps of the method. The foregoing description and drawings merely explain and rate the invention, and the invention is not limited thereto, except insofar as the claims are so limited.

Those skilled in the art that have the disclosure before them will be able to make modifications and variations therein without departing from the scope of the invention.

Forms of the Invention include: 1. An improved antioxidant ition for animal feed and constituents of animal feed sing lipid soluble tea catechins substituted for some or all of one or more antioxidant compounds in an unimproved antioxidant composition. 2. A composition as defined in form 1, wherein the unimproved antioxidant composition comprises tocopherols some or all of which are substituted with lipid e tea catechins. 3. A ition as defined in form 1, wherein the unimproved antioxidant composition comprises a water soluble tea extract some or all of which are tuted with lipid e tea catechins. 4. A composition as defined in form 1, further comprising a carnosic acid-containing extract of a Lamiaceae spp. plant tuted for some or all of one or more antioxidant compounds in an unimproved antioxidant composition.

. A composition as defined in form 4, wherein said Lamiaceae spp. plant is selected from the group ting of basil, mint, rosemary, sage, savory, marjoram, oregano, thyme and lavender. 6. An improved antioxidant composition for animal feed and constituents of animal feed comprising a rosmarinic acid-containing extract of a Lamiaceae spp. plant substituted for some or all of one or more antioxidant compounds in an unimproved antioxidant ition.

AH26(11310462_1):JIN 7. A composition as defined in form 6, wherein the unimproved antioxidant composition comprises tocopherols some or all of which are substituted with a rosmarinic acid-containing extract of a eae spp. plant. 8. A composition as defined in form 6, further comprising a carnosic acid-containing extract of a Lamiaceae spp. plant tuted for some or all of one or more antioxidant compounds in the unimproved antioxidant composition. 9. A composition as defined in form 8, wherein said Lamiaceae spp. plant is selected from the group consisting of basil, mint, rosemary, sage, savory, marjoram, oregano, thyme and lavender.

. A method of improving an antioxidant composition, comprising the step of tuting some or all of one or more idant compounds in the ition with lipid e tea catechins. 11. A method as defined in form 10, wherein the antioxidant composition comprises tocopherols some or all of which are substituted with lipid soluble tea catechins. 12. A method as defined in form 1, wherein the unimproved idant composition comprises a water soluble tea extract some or all of which are substituted with lipid soluble tea catechins. 13. A method as defined in form 10, further comprising a carnosic acid-containing extract of a Lamiaceae spp. plant substituted for some or all of one or more antioxidant compounds in an unimproved antioxidant composition. 14. A method as defined in form 13, wherein said Lamiaceae spp. plant is selected from the group ting of basil, mint, rosemary, sage, savory, marjoram, oregano, thyme and lavender.

. A method for improving an antioxidant ition for animal feed and constituents of animal feed comprising the step of substituting a rosmarinic acid-containing extract of a Lamiaceae spp. plant substituted for some or all of one or more antioxidant compounds in the antioxidant composition.

AH26(11310462_1):JIN 16. A method as defined in form 15, wherein the antioxidant composition comprises tocopherols some or all of which are substituted with a rosmarinic acid-containing extract of a Lamiaceae spp. plant. 17. A method as d in form 15, further sing a carnosic acid-containing extract of a Lamiaceae spp. plant substituted for some or all of one or more antioxidant compounds in the antioxidant ition. 18. A method as defined in form 17, wherein said Lamiaceae spp. plant is selected from the group consisting of basil, mint, rosemary, sage, savory, marjoram, oregano, thyme and lavender. 19. A method for ting animal fat from ion during rendering, comprising adding an antioxidant composition comprising lipid soluble tea catechins to the fat prior to or while being processed or held at temperatures above ambient.

. A method as defined in form 19, further comprising a carnosic acid-containing extract of a Lamiaceae spp. plant.

AH26(11310462_1):JIN I/WE

Claims (6)

CLAIM :

1. Animal feed or a constituent of animal feed comprising a erol and a rosmarinic acid-containing extract of a Lamiaceae spp. plant.

2. The animal feed or constituent of animal feed as defined in claim 1, further comprising a carnosic acid-containing extract of a Lamiaceae spp. plant.

3. The animal feed or constituent of animal feed defined in claim 1 or claim 2, n said Lamiaceae spp. plant is selected from the group consisting of basil, mint, rosemary, sage, savory, marjoram, oregano, thyme and lavender.

4. The animal feed or constituent of animal feed as defined in any one of claims 1 to 3 whereby the composition includes between 10 ppm and about 250 ppm of tocopherol.

5. The animal feed or constituent of animal feed as d in claim 4 whereby the composition includes between 10 ppm and about 240 ppm of tocopherol.

6. The animal feed or constituent of animal feed as defined in any one of claims 1 to 5 whereby the composition includes at least 50 ppm inic acid-containing extract. Kemin ries, Inc. By the Attorneys for the Applicant SPRUSON & FERGUSON Per: AH26(13851987_1):MXC