MIXTURE COMPRISING HYDROGENATED SOYBEAN OIL AND THYMOL

Abstract

The present invention relates to use of preferably fully hydrogenated soybean oil for manufacturing a particulate feed additive that is resistant to heat induced caking The mixture of the invention comprises hydrogenated soybean oil and thymol, wherein the weight ratio between hydrogenated soybean oil and thymol is from 10:1 to 1:1 and/or wherein the mixture comprises 5-30 weight-% of thymol, based on the total weight of the mixture. Preferably, the mixture is shaped as particles by spray chilling. The particles are then used as a feed additive. The feed additive may be a premix.

Claims

1. Mixture comprising hydrogenated soybean oil and thymol, wherein the weight ratio between hydrogenated soybean oil and thymol is from 10:1 to 1:1, and wherein the mixture comprises 5-30 weight-% of thymol, based on the total weight of the mixture.

2. Mixture according to claim 1, wherein said mixture further comprises 0.1 20 weight-%, preferably 1-15 weight-%, more preferably 1-10 weight-% and most preferably 1-8 weight-% of at least one terpene, based on the total weight of the mixture, and wherein said at least one terpene is liquid at room temperature.

3. Mixture according to claim 1, wherein said mixture comprises: fully hydrogenated soybean oil, 5-30 weight-%, preferably 10-20 weight-% and most preferably 12 28 weight-% thymol, based on the total weight of the mixture, 0.1-20 weight-%, preferably 1-15 weight-%, more preferably 1-10 weight-% and most preferably 1-8 weight-% eugenol, based on the total weight of the mixture and optionally at least one alkaloid being preferably piperine, wherein the weight ratio between fully hydrogenated soybean oil and thymol is from 10:1 to 1:1, preferably from 8:1 to 2:1, more preferably from 7:1 to 3:1 and most preferably from 6:1 to 5:1.

4. Mixture according to claim 1, wherein said mixture further comprises at least one auxiliary compound, and wherein said at least one auxiliary compound is preferably silicic acid, calcium carbonate, stearic acid, glycine and/or starch.

5. Particles comprising or consisting of the mixture according to claim 1.

6. Particles according to claim 5, wherein said particles are obtainable by a method comprising the steps: i. providing a mixture that comprises thymol, optionally eugenol, at least one auxiliary compound and molten hydrogenated soybean oil; ii. cooling the mixture provided in step i) by spraying said mixture into a cooling medium.

7. Premix comprising the mixture according to claim 1.

8. Premix according to claim 7, wherein one kilogram of said premix comprises 0.1 g to 10 g.

9. Food or feed, comprising the mixture according to claim 1. cm 10. Feed according to claim 9, wherein one ton of said feed comprises 1 g to 100 g of the mixture.

11. Method of manufacturing particles comprising hydrogenated soybean oil and thymol, said method comprising the steps: i. providing the mixture according to claim 1, wherein the mixture has a temperature of at least 65° C.; ii. cooling the mixture provided in step i) by spraying said mixture into a cooling medium.

12. Method according to claim 11, wherein the mixture provided in step i) has a temperature of at least 67° C., preferably at least 70° C., more preferably at least 72° C., even more preferably at least 75° C. and most preferably at least 80° C.

13. Use of fully hydrogenated soybean oil for manufacturing a particulate feed additive that is resistant to heat induced caking.

14. Use according to claim 13, wherein said particulate feed additive comprises thymol and optionally at least one terpene that is liquid at room temperature.

15. Use according to claim 14, wherein the weight ratio between fully hydrogenated soybean oil and thymol is from 10:1 to 1:1, and/or wherein the weight ratio between thymol and the at least one terpene is from 100:1 to 1.5:1.

Description

FIGURES

[0102] FIG. 1a shows the Differential Scanning calorimetry (DSC) thermogram of hydrogenated palm oil (HPO). DSC is a thermoanalytical technique in which the difference in the amount of energy required to increase the temperature of a sample respective to its environment is measured as a function of temperature. On the x-axis, the temperature is shown in ° C. On the y-axis, energy flow is shown (normalized, i.e. Watt/g of the composition). Negative energy flow indicates endothermic processes (e.g. melting). In FIGS. 1 a to 3, minima are endothermic peaks. Thus, the DSC thermogram in FIG. 1a shows two endothermic peaks. Positive energy flow corresponds to exothermic processes. In the FIGS. 1 a to 3, maxima are exothermic peaks. Thus, the DSC thermogram in FIG. 1a shows one exothermic peak.

[0103] FIG. 1b shows the DSC thermogram of fully hydrogenated soybean oil (FHSO). The DSC thermogram of FIG. 1b also shows two endothermic peaks. However, in comparison to the DSC thermogram shown in FIG. 1 a corresponding to the thermogram of fully hydrogenated palm oil (HPO), the two endothermic peaks of FHSO appear at higher temperatures.

[0104] In FIG. 2, DSC thermograms of the samples of comparative Example 2 are shown: sample 1a (HPO), sample 2 (ThyHPO) and sample 3 (EugThyHPO). ThyHPO is a mixture of thymol (Thy) and hydrogenated palm oil (HPO). FIG. 2 shows that ThyHPO melts at a lower temperature than HPO as such. EugThyHPO is a mixture of eugenol (Eug), thymol (Thy) and hydrogenated palm oil (HPO). FIG. 2 shows that EugThyHPO melts at an even lower temperature. The effects shown in FIG. 2 have some similarities with a phenomenon known as freezing-point depression.

[0105] In FIG. 3, DSC thermograms of the samples of Example 3 are shown: sample 1 b (FHSO), sample 4 (ThyFHSO) and sample 5 (EugThyFHSO). ThyFHSO is a mixture of thymol (Thy) and fully hydrogenated soybean oil (FHSO). FIG. 3 shows that the melting temperature of ThyFHSO is as high as the melting temperature of FHSO as such. This is surprising: when mixing thymol with FHSO, the freezing-point depression phenomenon does not show. EugThyFHSO is a mixture of eugenol (Eug), thymol (Thy) and fully hydrogenated soybean oil (FHSO). FIG. 3 shows that not even the addition of an oily liquid (eugenol) lowers the melting temperature of FHSO as such. Therefore, FHSO can be used for manufacturing a particulate feed additive that is resistant to heat induced caking.

EXAMPLES

Example 1

[0106] In Example 1, the melting points of hydrogenated palm oil (HPO) and fully hydrogenated soybean oil (FHSO) were determined by Differential Scanning calorimetry using a Discovery DSC (TA Instruments, Waters GmbH, Eschborn). Determination of melting points in Example 1 is from the 2.sup.nd heating cycle at 5° C. per minute from −10° C. to 90° C. Melting point was determined by the peak temperature T.sub.p (cf. G. Höhne, H. Cammenga, W. Eysel, E. Gmelin and W. Hemminger, “The Temperature Calibration of Scanning calorimeters,” Thermochimica Acta, vol. 160, pp. 1-12, 1990). The results are shown in FIG. 1a (HPO) and FIG. 1b (FHSO).

[0107] Both samples, HPO (sample 1a) and FHSO (sample 1b), display two melting peaks corresponding to different fatty acid compositions and chain length within the triacylglyceride (TAG). Possibly, the first melting peak relates to C16:0 chains within the TAG and alpha crystals while the second melting peak might relate to C18:0 chains within the TAG and beta crystals. Unless the second melting peak has been reached, the corresponding product (HPO or FHSO) is not fully liquid. The exothermic peak separating the two endothermic peaks may relate to melt-mediated transformation of crystals.

[0108] The analysis of the data shown in FIGS. 1a and 1b is given in below

TABLE-US-00002 TABLE 1 Table 1 Sample Endothermic peak (° C.) HPO 46.9 (C16:0) 57.0 (C18:0) FHSO 52.9 (C16:0) 61.5 (C18:0)

[0109] FHSO shows slightly higher melting temperatures than HPO. This might be related to differences between the respective fatty acid compositions: FHSO comprises less C16:0 triacylglycerides than HPO but more C18:0 triacylglycerides than HPO (R. Tieko Nassu and L. A. Guaraldo Goncalves, “Determination of melting point of vegetable oils and fats by differential scanning calorimetry (DSC) technique,” Grasas y aceotes, pp. 16-22, 1992 and I. V. J. R. G. L. R. M. Teles dos Santos, “Thermal properties of palm stearin, canola oil and fully hydrogenated soybean oil blends: Coupling experiments and modeling,” Journal of Food Engineering, vol. 185, pp. 17-25, 2016).

Comparative Example 2

[0110] In Example 2, two samples were prepared by the following process: [0111] 1. Melting of hydrogenated palm oil (HPO) in a 75° C. water bath. [0112] 2. Addition of thymol (sample 2), or thymol and eugenol (sample 3), one after the other while stirring at 200 rpm. [0113] 3. Speed up of stirring (500 rpm) and mix for 3 min. [0114] 4. Cool down slowly at room temperature.

[0115] During steps 2-3 of the preparation process, temperature was set to 75° C. No separation of oils was observed during cooling step 4 (i.e. the surface remained “dry”). After cooling, the composition was grounded, and samples were taken for DSC analysis. All ingredients are commercially available. Thymol (purity: 99%) was purchased at VWR Chemicals, eugenol (purity: 99%) at Merk KGaA.

[0116] The composition of samples 2 and 3 as prepared in Example 2 is shown in below TABLE 2. In comparison, the composition of sample 1a of Example 1 is also shown in Table 2.

TABLE-US-00003 TABLE 2 sample 1a sample 2 sample 3 HPO ThyHPO EugThyHPO hydrogenated palm oil (HPO) 5 g 17.1 g 15.59 g thymol 0 g  2.9 g  2.91 g eugenol 0 g    0 g  1.5 g total weight 5 g   20 g   20 g weight ratio HPO:thymol n/a 5.9:1 5.4:1

[0117] For each of the samples, a melting curve was measured by Differential Scanning calorimetry, using a Discovery DSC (TA Instruments, Waters GmbH, Eschborn). Melting points were determined as described in Example 1. The obtained the DSC thermograms are shown in FIG. 2.

[0118] FIG. 2 shows that the addition of thymol to HPO merges the peaks of HPO into one endothermic peak. Thereby, the merged peak appears at lower temperature (51.9° C.) than the 2.sup.nd endothermic peak of pure HPO (57.0° C.). 51.9° C. is a temperature that may be reach in a closed truck standing in the sun during summer. Therefore, the formation of lumps (caking) during transportation cannot be excluded when using HPO for manufacturing a particulate feed additive. The risk of heat induced caking becomes ever higher when both, thymol and eugenol are admixed to HPO (sample 3): the merged peak of such mixture appears at an even lower temperature (49.8° C.).

Example 3

[0119] In Example 3, the approach of Example 2 was repeated. In Example 3, however, fully hydrogenated soybean oil (FHSO) was used instead of HPO.

[0120] The composition of samples 4 and 5 as prepared in Example 3 is shown in below TABLE 3. In comparison, the composition of sample 1b of Example 1 is also shown in Table 3.

TABLE-US-00004 TABLE 3 sample 1b sample 4 sample 5 FHSO ThyFHSO EugThyFHSO fully hydrogenated soybean oil 5 g  17.1 g 15.59 g (FHSO) thymol 0 g  2.91 g  2.91 g eugenol 0 g    0 g  1.51 g total weight 5 g 20.01 g 20.01 g weight ratio n/a 5.9:1 5.4:1 FHSO:thymol

[0121] For each of the samples, a melting curve was measured by Differential Scanning Calorimetry as described in Example 2. The obtained the DSC thermograms are shown in FIG. 3.

[0122] FIG. 3 shows that the addition of thymol to FHSO merges the peaks of pure FHSO into an endothermic peak. This is similar to FIG. 2. However, apart from this similarity, there are major differences.

[0123] When adding thymol to FHSO, an exothermic peak is observed from 12° C. to about 33° C. This exothermic peak appears regardless whether or not eugenol has also been added. This possibly indicates a crystal reconfiguration which does not take place in case of HPO (cf. FIG. 2).

[0124] More importantly, and very surprising, the risk of heat induced caking is not increased or is even decreased when thymol (sample 4) or thymol and eugenol (sample 5) are admixed to FHSO: the endothermic peaks of the respective mixtures appear at about the same temperature as the 2.sup.nd endothermic peak of FHSO (61.5° C.) as such. An overview of the results of Examples 2 and 3 is given in below TABLE 4.

TABLE-US-00005 TABLE 4 1.sup.st 2.sup.nd Δ to respective 2.sup.nd endothermic endothermic endothermic peak Sample peak (° C.) peak (° C.) (° C.) FHSO 52.9 61.5 ThyFHSO 62.1 0.6 EugThyFHSO 61.8 0.3 HPO 46.9 57.0 ThyHPO 51.9 −5.1 EugThyHPO 49.8 −7.2

[0125] In case of two endothermic peaks, a fully melted composition is not obtained until the temperature of the 2.sup.nd peak has been reached. Therefore, the melting point of ThyFHSO (62.1° C.) is about 10° C. higher than the melting point of ThyHPO (51.9° C.) whereas the melting point of FHSO (61.5° C.) is only about 4.5° C. higher than the melting point of HPO (57° C.). This is surprising.

[0126] This surprising effect is even more pronounced if both, thymol and eugenol are added: the melting point of EugThyFHSO (61.8° C.) is about 12° C. higher than the melting point of EugThyHPO (49.8° C.) whereas the melting point of FHSO (61.5° C.) is only about 4.5° C. higher than the melting point of HPO (57° C.).

[0127] The likelihood that a temperature of 61.8° C. (cf. EugThyFHSO) is reached during transportation is lower than the likelihood that a temperature of 49.8° C. (cf. EugThyHPO) is reached in a closed truck during summer. Therefore, heat induced caking can be prevented or at least reduced when using FHSO instead of HPO for manufacturing a particulate feed additive.

Example 4

[0128] Particles comprising the mixture of the invention were manufactured as follows:

[0129] Molten fully hydrogenated soybean oil was mixed with thymol, eugenol and selected auxiliary compounds. To obtain particles, the hot, liquid mixture was cooled by spraying (spray chilling). Organoleptic inspection of the obtained particles confirmed a reduced smell.

[0130] The thus obtained particles were a flowable powder. The powder was then stored in a climatic chamber for 3 days in conditions of 52.5° C. and relative humidity (rH) of 60%. After the elapsed period, the powder was still flowable. No lumps could be observed. Example 4 shows that particles of the invention are resistant to heat induced caking.

[0131] The powder of Example 5 can be used to prepare a premix. Feed comprising the thus prepared premix may then be fed to broilers or other animals.

Comparative Example 5

[0132] In Example 5, particles were manufactured as described in Example 4. However, instead of fully hydrogenated soybean oil, hydrogenated palm oil was used in Example 5. The thus manufactured particles were a flowable powder. The powder of Example 5 was then also stored in a climatic chamber for 3 days in conditions of 52.5° C. and rH of 60%, similar to Example 4. However, after the elapsed period, the powder of Example 5 was no longer flowable. Instead, the previously flowable powder has melted together and has become one large, solid object. Thus, the particles of Example 5 are prone to heat induced caking.

[0133] After having been exposed to a temperature of 52.5° C., the powder prepared in Example 5 could no longer be used to prepare a premix. Large, solid agglomerates are useless and thus, must be discharged.

Example 6

[0134] On a sunny day in the late afternoon, the temperature was measured in a truck on a parking lot in Italy. Air-conditioning and engine had been switched off. The loading area of the truck was covered, and all windows were closed. When the temperature was measured, the truck had been on the parking lot for about 8 hours.

[0135] A temperature of about 49° C. was measured inside the truck. In the previous examples, the melting temperature of EugThyFHSO has been determined as 61.8° C. and would therefore resist the temperature measured in the truck of Example 6.

Example 7

[0136] In Example 7, antimicrobial activity of thymol, eugenol and a combination of thymol and eugenol was evaluated against pathogenic bacteria. Thymol and eugenol were purchased from Sigma-Aldrich (St. Louis, MO, USA). They were stored at 4° C. before use.

[0137] Bacterial strains: Three strains of pathogenic bacterial, E. coli K88+, S. choleraesuis and Cl. perfringens obtained from China Veterinary Culture Collection Center were used to determine the antimicrobial activity of thymol, eugenol and/or thymol. E. coli K88+and S. choleraesuis were aerobic and isolated from the gastrointestinal tract of swine, Cl. perfringens was anerobic and isolated from poultry. The three strains were kept in broth with 25% glycerol at −80° C.

[0138] Antimicrobial activity of thymol and eugenol: The minimum inhibitory concentration (MIC) values of thymol and eugenol, respectively, were determined using two-fold broth dilution method. The compounds were dissolved in analytical grade ethanol and serially diluted to yield various concentrations, typically in the range of 6.03-368.17 mmol/L. The bacterial suspensions were measured at OD600.sub.nm and standardized to a concentration of 10.sup.5-10.sup.6 CFU/mL with the culture broth. Aliquots of 150 μL of each bacterial broth were pipetted into the wells of a 100-well microtiter plate and 3.14 μL of eugenol or thymol concentration was, respectively, added into the wells followed by adding 150 pl of bacterial suspensions to give a final ethanol concentration at 1%. A blank control well contained bacterial broth and suspensions, and 3.04 pL of ethanol instead of eugenol or thymol. The plate was incubated with shaking at 37° C. with the Bioscreen C system (Labsystem, Helsinki, Finland). The growth of bacterial was measured by reading the OD600nm at 30 min intervals for 24 hr and kinetic curves were analysed. The MIC was considered as the lowest concentration showing no growth of bacterial. All the tests were carried out in triplicate, and mean value was calculated. All the procedures with Cl. perfringens were carried out under anaerobic conditions.

[0139] Antimicrobial activity combination: thymol and eugenol were assessed in combination to determine their activity against E. coli K88+as described previously. The kinetic curves were analysed by Origin 2017 calculating lag phase (A), which was selected as criteria for comparison of antimicrobial efficacy.

[0140] The results of Example 7 are shown in TABLES 5 and 6.

[0141] For Escherichia coli K88+and Salmonella choleraesuis the ranking of antimicrobial performance based on MIC values was: thymol >eugenol. The particles of the present invention comprise thymol and show therefore excellent antimicrobial performance.

[0142] The duration of the lag phase (A) is criteria for antimicrobial efficacy. During lag phase, the cells adapt to a new environment. Lag phase is then followed by the log phase, in which population grows in a logarithmic fashion. The grown cells are harmful, and thus, the longer the lag phase, the better. The data in Table 6 shows that combination of thymol and eugenol results in a longer lag phase than the same amount of thymol alone or eugenol alone. A preferred embodiment of the invention relates to a mixture comprising both, thymol and eugenol. The product of said preferred embodiment is particularly effective to combat E. coli K88+.

TABLE-US-00006 TABLE 5 Minimum inhibitory concentration (MIC) values of thymol and eugenol against Escheria coli K88.sup.+, Salmonella cholerasuis and Clostridium perfingens MIC values (mmol/L) Escheria Salmonella Clostrisium compound coli K88.sup.+ choleraesius perfringens Thymol 1.5 1 1 Eugenol 2.5 2.5 4

TABLE-US-00007 The Lag phase (λ) of thymol and eugenol individually or in combination against Escheria coli K88.sup.+1 Essential Dosage, Dosage, oil mmol/L λ mmol/L λ Individually Thymol 0.5 1.65 0.25 1.66 Eugenol 0.5 1.91 0.25 1.77 Combination Thymol + Eugenol 0.25 + 0.25 1.99