Preparation of Water-Dispersible Rosemary Antioxidant and Evaluation of Its Antioxidant Effect in Minced Pork Products

 Abstract:In this paper, a high performance liquid chromatographic method was developed for the simultaneous determination of five components (rhamnol, rhamnolic acid, rosmarinic acid, oleanolic acid and ursolic acid) in the extract of rosemary, and the preparation conditions of the water-dispersible rosemary antioxidant were optimized, and the antioxidant effect of the antioxidant was measured when it was added into the minced pork. The results showed that the optimal conditions for the preparation of the water-dispersible rosemary antioxidant were as follows: polyglycerol fatty acid ester and Tween 80 in the ratio of 1:5 (m/m), oil-water phase in the ratio of 4:6 (m/m), emulsification at 2800 r/min, and the particle size of the antioxidant was 318.83 nm. The addition of 0.03% water-dispersible rosemary antioxidant to the minced pork was significantly (P<0.01) more efficient than that of other antioxidants. The addition of 0.03% water-dispersible rosemary antioxidant to minced pork could significantly (P＜0.05) slow down the decrease of L ∗ and a ∗, inhibit the increase of colony counts, delay oxidation and spoilage, and extend the shelf-life of minced pork by 4 d compared with that of the unadded group.

 

Rosemary (Rosmarinus oficinalis) belongs to the Labiatae family of aromatic plants, native to the Mediterranean region. Rosemary extracts are divided into two categories: fat-soluble and water-soluble extracts, the former mainly containing rhamnetinic acid and rhamnetinol, and the latter mainly containing rosmarinic acid, which have good antioxidant activity[1-2] . As an antioxidant, the fat-soluble extract of rosemary has the advantages of naturalness, high efficiency and high temperature resistance, and is one of the potential natural antioxidants [3-6]. The fat-soluble extract of rosemary also has some bacteriostatic activity, therefore, it can be used as both antioxidant and preservative in food [7-8].

 

Pork is rich in fat and protein, and after being made into minced pork, the water content is high, the original tissue structure is destroyed, and it is very susceptible to light, temperature, water, air and microorganisms, leading to rancidity and spoilage, affecting the quality and shelf life of pork and minced pork[9-12] . Currently, synthetic antioxidants such as butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT), and tert-butylated hydroquinone (TBHQ) are commonly added to prevent oxygenation of minced pork, and preservatives are also needed to inhibit microbial proliferation [13-15]. However, the safety of synthetic antioxidants has always been a concern for consumers, and the approval and limit of various synthetic antioxidants in the United States, the European Union, Japan, and China are different, while at the same time, food products using natural antioxidants are considered safer and healthier by consumers, and are more popular among consumers [16-18]. Therefore, rosemary antioxidant, as a natural antioxidant, can prevent the oxidation of pork and inhibit the proliferation of microorganisms[19-20] .

 

The antioxidant effect of rosemary extract in meat products was reported to be significant, and it could help to maintain the stability of the color and luster of meat products and inhibit the growth of microorganisms. Wang Zhengrong et al [21] found that chitosan coating with rosemary extract had a compound freshness preservation effect on pork patties, which could inhibit fat oxidation, protein degradation and microbial growth of pork patties to a certain extent, and had a certain color protection effect, which could extend the shelf life of refrigerated pork patties to about 15 days. Feng et al [23] found that the addition of rosemary extract had a favorable effect on the TVB-N value, PV value, TBA value, pH and microbial count of chicken breast meat, and had a certain color protection effect. However, the fat-soluble components of rosemary extract are not easy to be dispersed evenly in meat products. Therefore, in this study, it was proposed to make the fat-soluble rosemary extract into water-dispersible form by emulsification and homogenization and to optimize the preparation process. The aqueous-dispersible rosemary extract was added into minced pork to study its effect on the microbial and oxidative stability of minced pork.

 

1 Materials and Methods

1.1 Materials and instruments

Rosemary fat-soluble extract of Henan Jiashang Agricultural Science and Technology Development Company Limited; raw meat (lean pork hind leg, backfat) a supermarket in Beijing; salt China Salt Beijing Salt Industry Company; edible monosodium glutamate Henan Lianhua monosodium glutamate Co. (purity ≥ 98%) Chengdu Manstar Biotechnology Co., Ltd; oleanolic acid standard (purity ≥ 98%), ursolic acid standard (purity ≥ 98%) Sigma Biotechnology Corporation; methanol (chromatographic purity), phosphoric acid (chromatographic purity) Beijing Mairenda Technology Company Limited; polyglycerol fatty acid ester Shandong Konost Biotechnology Co. Tween 80, thiobarbituric acid, 1,1,3,3-tetraethoxypropane, Shanghai Yuanye Bio-technology Co.

 

LC1200 Liquid Chromatography Instrument, Agilent, USA; KQ-100E Ultrasonic Cleaner, Kunshan Ultrasonic Instrument Co., Ltd; HC-2062 High-Speed Freeze Centrifuge, Anhui Zhongjia Scientific Instrument Co. MPH-M3 High Pressure Homogenizer Shanghai Mogul Machinery Co., Ltd. ;Zetasizer Nano ZS90 ZETA Potentiostat Malvern Instruments Co. Shanghai Bo Xun Industry Co.

 

1.2 Experimental methods

1.2.1 Determination of the main components of rosemary fat-soluble extract by high performance liquid chromatography

1.2.1.1 Sample handling    

5.0 mg of rosemary fat-soluble extract was weighed and dissolved in 5 mL of aqueous methanol phosphate solution (methanol:0.2% phosphate solution = 88:12, V/V), and then ultrasonicated for 10 min at room temperature.

The samples were filtered through 0.22 μm microporous membrane and analyzed by high performance liquid chromatography (HPLC).

 

1.2.1.2 Chromatographic methods    

The chromatographic column was a Waters C18 column (2.5 mm×250 mm, 5 μm). The mobile phase was methanol:0.2% phosphoric acid solution = 88:12 (V/V) with isocratic elution at a flow rate of 1.0 mL/min, and the peak intensities of 210 (oleanolic acid, ursolic acid), 230 (rhamnol, rhamnolic acid) and 330 nm (rosmarinic acid) were detected respectively. The column temperature was 25 ℃. The sample volume was 10 μL.

 

1.2.1.3 Preparation of standard curves    

Weigh 1.0 mg of the standards of Sage Phenol, Sage Acid, Rosmarinic Acid, Oleanolic Acid and Ursolic Acid respectively, mix the above five standards, dissolve them in 10 mL of mobile phase solution, and then dilute them to different concentrations for detection by HPLC.

 

1.2.1.4 Precision test The maximum concentration (100 μg/mL) of the mixed standard solution was taken, and the sample was injected continuously six times according to the above chromatographic method. The peak area of each substance was recorded and the relative standard deviation (RSD) was calculated.

 

1.2.1.5 Repeatability experiments    

Six batches of rosemary fat-soluble extracts were prepared at the same concentration, and the samples were injected according to the above chromatographic conditions, and the peak area of each substance was recorded and the RSD was calculated.

 

1.2.1.6 Stability experiments    

The maximum concentration of rosemary fat-soluble extract was aspirated and analyzed by HPLC at 0, 4, 8, 12, 16 and 24 h. The peak areas of the five substances were recorded and the RSDs were calculated at these six times.

 

1.2.2 Preparation of water-dispersible rosemary antioxidants    

Weigh 7.5 g Polyglycerol fatty acid ester (PGFE), add a certain amount of Tween 80, add ultrapure water to 800 g, ultrasonic at 60 ℃ for 10 min, so that it is fully dissolved, and then cooled to room temperature to obtain the aqueous phase components; Weigh 5.0 g of rosemary fat-soluble extract, add 50 mL of 60% ethanol, and then add medium chain triglycerides (MCT) to 200 g, stirred at room temperature for 10 min, to obtain the oil phase components. Add Medium chain triglycerides (MCT) to 200 g, and stir at room temperature for 10 min to fully dissolve the oil phase. Put a certain amount of oil phase along the wall of the cup in

The product was slowly added into the aqueous phase within 5 min, and emulsified by a shear emulsifier at a certain rotational speed, and continued to shear for 8 min to form a crude emulsion, which was then homogenized by a high pressure homogenizer at 20 MPa for 3 min to form a water-dispersed rosemary antioxidant.

 

1.2.3 Determination of Particle Size, Particle Size Distribution Index and Zeta Potential    

The 1.2.2 water-dispersible rosemary antioxidant was diluted 100 times and the particle size, particle size distribution index (PDI) and zeta potential were determined using a Malvern Zetasizer Nano ZS90 Zeta Potentiometer.

 

1.2.4 Determination of viscosity    

The viscosity of water dispersible rosemary antioxidant was determined by rheometer.

 

1.2.5 Measurement of light absorption stability    

Dilute 0.1 mL of water-dispersible rosemary antioxidant to 5 mL and measure the absorbance at 520 nm as T0. Take 20 mL of water-dispersible rosemary antioxidant in a centrifuge tube, heat it in a water bath at 60 ℃ for 1 h. Take 0.1 mL of the upper solution and dilute it to 5 mL, then determine the absorbance and record it as T1. The absorbance stability K was calculated as follows

K = T0/T1

 

1.2.6 One-way experiments    

The water-dispersible rosemary antioxidant was prepared by the method in 1.2.2. The preparation conditions were fixed at 2∶8 oil-water phase ratio and 2200 r/min emulsification speed to investigate the effects of different compounding ratios (1∶2, 1∶3, 1∶4, 1∶5, 1∶6, m/m) of PGFE and Tween 80 on the preparation of water-dispersible rosemary antioxidants. The effects of different oil-water phase ratios (1∶9, 2∶8, 3∶7, 4∶6, m/m) on the preparation of water-dispersed rosemary antioxidants were investigated at 2200 r/min. The effects of different emulsification speeds (1600, 2200 and 2800 r/min) on the preparation of water-dispersed rosemary antioxidants were examined by fixing the preparation conditions as 1∶3 PGFE-Twain 80 ratio and 2∶8 oil-water phase ratio. The effects of different emulsification speeds (1600, 2200, 2800 r/min) on the preparation of water-dispersed rosemary antioxidants were investigated. The effects of various variables on the preparation of water-dispersed rosemary antioxidants were investigated in terms of particle size, PDI, zeta potential, viscosity and light-absorbing stability.

 

1.2.7 Orthogonal tests    

The factors and levels of the orthogonal test were determined through the single-factor experiment, and the optimal three levels of the single factor were selected for the orthogonal test. According to Stokes' formula, the particle size and the stability of the water-dispersed rosemary antioxidant were completely positively correlated, which could well characterize the stability of the system. Therefore, the particle size was chosen as the main optimization index, and the orthogonal table of L9 (34) was used to optimize the three factors, namely, the ratio of the PGFE and Tween 80, the ratio of the oil and water phases, and the emulsification speed, which influenced the effect of the preparation of water-dispersed rosemary antioxidant. The three factors affecting the preparation effect of water-dispersible rosemary antioxidant were optimized by orthogonal test.

 

1.2.8 Application of water-dispersible rosemary antioxidants

 

1.2.8.1 Preparation of minced pork    

Weigh 500 g of lean pork hind leg and 100 g of pork backfat, remove the visible fascia, wash and split them, and then mince them. Then 18 g corn starch, 7 g salt, 0.2 g monosodium glutamate and 10 g distilled water were weighed, mixed well and added to the meat, and then divided into three groups according to the mass: the blank control group (CK group, without the addition of rosemary fat-soluble extract) and the two treatment groups (R0.01 group, R0.03 group) with the addition of 0.01% and 0.03% of the water-dispersible rosemary extract, respectively, each divided into 10 portions, of which 5 portions were stored at 0 ℃, and recorded. Each group was divided into 10 portions, of which 5 portions were stored at 0 ℃, recorded as Group A (CK1, R0.01-1, R0.03-1), and the other 5 portions were stored at 10 ℃, recorded as Group B (CK2, R0.01-2, R0.03-2). The samples were taken out on the 0th, 1st, 3rd, 5th and 7th day for the determination of TBA, color and volatile saline nitrogen content, respectively.

 

1.2.8.2 Determination of TBA value    

Determine the TBA value according to the second method of spectrophotometric method in GB 5009.181-2016 "Determination of Malondialdehyde in Food".

 

1.2.8.3 Determination of the color of ground pork    

The color change of ground pork during storage was determined using a Minolta CM-600d colorimeter. The colorimeter was calibrated with a standard plate (CIE 1931: Y = 94.0, x = 0.3156, y = 0.3156, y = 0.3156, y = 0.3156, y = 0.3156).

0.3321), the chromaticity (brightness L ∗ , redness a ∗ , yellowness b ∗ ) of the surface of the pork balls was measured by using a D65 light source, a measuring range of 8 mm in diameter and a viewing angle of 2°. Four evenly distributed locations on the ground pork were taken for the measurement, and the probe was held tightly to the surface of the meat during the measurement without leakage of light, and the values of brightness L ∗ , redness a ∗ , and yellowness b ∗ were recorded (the samples from the 10 ℃ control group were used as the standard samples).

 

1.2.8.4 Determination of Volatile Salt-Based Nitrogen Content      

Determination according to GB5009.228-2016 "Determination of Volatile Salt Nitrogen in Foods, National Standard for Food Safety".

 

1.2.8.5 Determination of the total number of colonies    

The same three experimental groups of minced pork were prepared according to the above recipe and process, and were incubated in the incubator at 0 and 10 ℃, and the total number of colonies was determined according to the time points in 1.2.8.1. The total number of colonies was determined according to GB 4789.2-2016 National Standard for Food Safety, Microbiological Examination of Food, Determination of Total Colony, and the results were expressed as the logarithmic value of the total number of colonies, lg (CFU/g).

 

1.3 Data processing

The experimental procedure was repeated three times, and the experimental data were expressed as ± S, and were collected by

One-way analysis of variance (ANOVA) and multiple comparisons were performed using SPSS Statistics 17.0 software, and the results of the orthogonal tests were analyzed using Minitab 17.0 software, and plotted using OriginPro 8.5.1 software.

 

2 Results and analysis

2.1 Determination of the main components of rosemary fat-soluble extract by high performance liquid chromatography

The mixed standards were detected by high performance liquid chromatography (HPLC), and the retention times of rosmarinic acid, rhamnetol, rhamnol, oleanolic acid and ursolic acid were 2.780, 4.708, 9.061, 19.668 and 20.631 min, respectively. 6 consecutive injections were made into the same sample to validate the precision of the method, and the RSDs of rosmarinic acid, rhamnetol, rhamnol, oleanolic acid and ursolic acid were 0.65%, 0.37%, 0.78%, 0.35% and 0.59%, which were less than 10% and met the methodological requirements. The RSDs of the peak areas of rosmarinic acid, salvinorinol, salvia divinorum, oleanolic acid and ursolic acid were 0.65%, 0.37%, 0.78%, 0.35%, 0.59%, which were less than 10%, in accordance with the methodological requirements, indicating that the precision of the instruments used was good.

 

Six batches of rosemary fat-soluble extracts with the same concentration were injected into the sample at the same time to verify the reproducibility of the method, and the RSDs of the peak areas of rosemarinic acid, rhamnol, rhamnolic acid, oleanolic acid and ursolic acid were 3.38%, 2.57%, 4.99%, 4.76% and 1.82%, respectively, which were less than 10%, and conformed to the requirements of the methodology, indicating that the reproducibility of the method was good.

 

The maximum concentration of rosemary fat-soluble extract solution was analyzed by HPLC at 0, 4, 8, 12, 16 and 24 h to verify the stability of the method, and the RSDs of the peak areas of rosemarinic acid, rhamnetol, rhamnolic acid, oleanolic acid and ursolic acid were 0.36%, 1.56%, 1.79%, 0.87%, 1.42%, which were less than 10%, respectively. The RSDs of the fat-soluble extracts of rosemary were less than 10%, which met the requirements of the methodology and indicated that the stability of the extracts was good at room temperature within 24 hours.

 

The results of precision, reproducibility and stability experiments showed that the HPLC method can simultaneously detect the contents of five components in rosemary extracts (salvinorin, salicylic acid, rosmarinic acid, zymosanic acid and ursolic acid) and the results are good.

 

2.2 Results of one-way experiments

2.2.1 Influence of the ratio of PGFE and Tween 80 on the antioxidant properties of water dispersible rosemary    

From Fig. 2, it can be seen that the particle size of the water-dispersed rosemary antioxidant showed a tendency of decreasing and then increasing with the increase of the proportion of PGFE and Twain 80, and the PDI value showed a tendency of decreasing and then increasing and then decreasing, and the particle size and the PDI value were the smallest with the ratio of 1∶4 (m/m); and the potential, viscosity and light-absorption stability of the water-dispersed rosemary antioxidant showed a certain significant (P＜0.05) change trend. The potential, viscosity and light absorption stability of the water-dispersed rosemary antioxidant showed some significant (P＜0.05) trends, and the light absorption stability was the best when the compounding ratio was 1∶4 (m/m), while the potential and viscosity were the highest when the compounding ratio was 1∶5 (m/m), but there was no significant difference between the 1∶4 (m/m) and the 1∶5 (m/m). When the five indexes were combined, the dispersion effect of the water-dispersed rosemary antioxidant was better when the PGFE:Tween 80 was 1:4 (m/m). Although the particle size, PDI and light absorption stability of 1:3 were better than that of 1:6, the potential and viscosity of PGFE:Tween 80 were worse than that of 1:6. Since the price of PGFE was about 3 times of Tween 80, the interval of 1∶4～1∶6 was chosen for the orthogonal experiments.

 

2.2.2 Influence of oil-water phase ratio on the properties of water-dispersed rosemary antioxidants    

From Fig. 3, it can be seen that the particle size, PDI value, potential and viscosity of aqueous dispersed rosemary antioxidant showed a tendency of decreasing and then increasing with the increase of the ratio of oil to water phase, the PDI value was the smallest when the ratio of oil to water phase was 3∶7 (m/m), and the smallest particle size of aqueous dispersed rosemary antioxidant was found when the ratio of oil to water phase was 2∶8 (m/m), but there was no significant difference between the ratio of oil to water phase and that of oil to water phase was 3∶7 (m/m). The potential and viscosity of the water-dispersed rosemary antioxidant were the highest at the oil-water phase ratio of 4:6 (m/m), but there was no significant difference between the 3:7 (m/m) and the water-dispersed rosemary antioxidant. With the increase of oil-water phase ratio, the light absorption stability of water-dispersed rosemary antioxidant showed a tendency of increasing and then decreasing, and the highest light absorption stability was found at the oil-water phase ratio of 3∶7 (m/m). When the oil-water phase ratio was 3:7 (m/m), the dispersion effect of water-dispersed rosemary antioxidant was better. Taking into consideration of the five indicators, the oil-water phase ratio of the orthogonal experiment was chosen to be 2:8, 3:7 and 4:6 for the subsequent investigation.

 

2.2.3 Effect of emulsification revolutions on the properties of water-dispersed rosemary antioxidants    

As shown in Fig. 4, with the increase of emulsification speed, the particle size, PDI value, potential and viscosity of the water-dispersed rosemary antioxidant showed a decreasing trend, and the particle size, PDI value and viscosity of the water-dispersed rosemary antioxidant were the smallest at 2800 r/min, while the particle size, PDI value and viscosity of the water-dispersed rosemary antioxidant were the smallest at 2800 r/min, and the particle size, PDI value and viscosity were the smallest at 2800 r/min.

At 2200 r/min, the potential value of water-dispersed rosemary antioxidant was the smallest, but there was no significant difference at 2800 r/min. With the increase of emulsification speed, the light absorption stability of water-dispersed rosemary antioxidant showed a gradual increase, and the highest light absorption stability of water-dispersed rosemary antioxidant was found at 2800 r/min. The emulsification speeds of 1600, 2200 and 2800 r/min were chosen to investigate the dispersion effect of the water-dispersed rosemary antioxidant.

 

2.3 Optimization results of the preparation process of water-dispersed rosemary antioxidant

The results of the optimized process for the preparation of water dispersible rosemary antioxidants are shown in Table 2.

As shown in Table 2, the main factors affecting the particle size of water-dispersed antioxidants are, in descending order, the ratio of oil and water phases, the number of emulsifying revolutions, and the proportion of emulsifiers. The smaller the particle size, the better the dispersing effect of the water-dispersed rosemary antioxidant, and the optimal conditions were A3 B2 C3, i.e., 4:6 (m/m) oil:water, 1:5 (m/m) PGFE and Tween 80, and 2800 r/min. The particle size of the water-dispersed rosemary antioxidant prepared under the optimal conditions was 318.83 nm.

 

2.4 Determination of effective components of water-dispersible rosemary antioxidants

The water-dispersible rosemary antioxidant prepared under the optimal conditions was purified by C18 solid-phase microextraction (SPME) column, and the content of its active ingredients was determined by HPLC method. The results showed that the prepared water-dispersible rosemary antioxidant mainly contained two kinds of constituents, sageol and sage acid, with the contents of 0.0398% and 0.0223%, respectively.

 

2.5 Effect of water-dispersible rosemary antioxidant on TBA value of minced pork

A water-dispersible rosemary antioxidant was added into the minced pork, and the changes of TBA values of minced pork with storage time were determined at different temperatures, and the results are shown in Fig. 6.

TBA content is an important index for evaluating the degree of fat peroxidation, and the degree of fat oxidation is an important factor affecting food quality. As the degree of fat oxidation deepens, the secondary products increase continuously and the TBA content also increases[24] . According to Fig. 6, TBA value increased with time at both temperatures, and TBA was higher at 10 ℃, indicating that low-temperature storage was able to reduce the TBA value of minced pork within the same storage time. After the addition of water-dispersible rosemary antioxidant, the TBA values were reduced. At 10 ℃, the effect of 0.01% water-dispersible rosemary antioxidant was not obvious with the extension of storage time, but 0.03% rosemary antioxidant could reduce the TBA value better; at 0 ℃, the effect of water-dispersible rosemary antioxidant was better, and the concentration of 0.03% had the best effect, which indicated that rosemary antioxidant could inhibit the oxidation of the minced pork.

 

2.6 Effect of water dispersible rosemary antioxidant on the color value of minced pork

The water-dispersible rosemary antioxidant was added into the minced pork to determine the changes of L ∗ , a ∗ and b ∗ values of minced pork with the storage time under different conditions, and the results are shown in Fig. 7.

As shown in Fig. 7, the L ∗ and a ∗ values of minced pork showed a decreasing trend throughout the storage period, and the decrease was faster at 10 ℃, while the b ∗ value did not change significantly. After 5 d of storage at 0 ℃, the L ∗ values of the treatment groups with the addition of water-dispersible rosemary antioxidant were significantly higher than those of the blank control group (P＜0.05), and the effect of the addition of 0.03% antioxidant was better (P＜0.05). After 3 d of storage, the a ∗ values showed the same trend. The Fe2+ contained in hemoglobin made the pork bright red, but with the extension of storage time, the Fe2+ was gradually oxidized, and the color of the pork would gradually become dark gray. As a result, the L ∗ and a ∗ values tend to decrease gradually. The addition of water-dispersible rosemary antioxidant significantly slowed down the decrease of L ∗ and a ∗ values (P＜0.05), which indicated that it had a certain protective effect on the color of minced pork.

 

2.7 Effect of water-dispersed rosemary antioxidant on TVB-N content in minced pork

A water-dispersible rosemary antioxidant was added into the minced pork to determine the changes of TVB-N value with storage time under different conditions, and the results are shown in Fig. 8.

TVB-N is an important indicator of the degree of spoilage of fresh meat and meat products, which is an alkaline nitrogenous substance produced by the decomposition of proteins contained in animal food by bacteria and enzymes, and the content of TVB-N increases with the increase of the degree of spoilage of meat and meat products[25] . As shown in Figure 8, the TVB-N values of all groups showed an increasing trend with the increase of storage time, and the TVB-N values of minced pork increased more rapidly at 10 ℃. The addition of water-dispersible rosemary antioxidant significantly inhibited the increase of TVB-N value (P＜0.05), and the TVB-N value of minced pork with 0.03% antioxidant was relatively lower (P＜0.05). According to GB 20799-2016, the TVB-N value of fresh meat should be less than 15 mg/kg. It can be seen that, at 10 ℃, the control group, 0.01% rosemary group and 0.03% rosemary group were spoiled on the 3rd, 5th and 7th d, respectively; and the control group, 0.01% rosemary group and 0.03% rosemary group were spoiled on the 7th, 11th and 13th d, respectively, at 0 ℃. In both groups, the shelf-life of minced pork with 0.03% rosemary could be extended by 4 d compared with that of the control group. Therefore, the addition of rosemary antioxidant could significantly extend the shelf-life of minced pork (P＜0.05).

 

2.8 Effect of water-dispersible rosemary antioxidant on the total bacterial count of pork mince

The antioxidant of water-dispersible rosemary was added into the minced pork, and the changes of the total number of colonies in the minced pork with the storage time under different conditions were determined, and the results are shown in Fig. 9. It can be seen that the total number of colonies increased rapidly with the prolongation of the storage time and the proliferation was more rapid at 10 ℃. The antibacterial effect of adding 0.01% rosemary antioxidant was not obvious, while the total number of colonies of minced pork with 0.03% rosemary antioxidant was significantly lower than that of the blank control group (P＜0.05), which showed a better antibacterial effect. According to GB 4789.2-2016, when the total number of colonies (lg(CFU/g)) was less than 6, it was fresh meat; when the total number of colonies (lg(CFU/g)) was more than 6, it was spoiled meat. As shown in Figure 9, at 10 ℃, the meat of all groups had been spoiled at the 3rd d, and at 0 ℃, the meat had been spoiled at the 7th d. The pork surimi with 0.03% antioxidant reached the index of spoiled meat at the 9th d. Therefore, the meat with 0.03% antioxidant had been spoiled at the 3rd d, and the pork surimi with 0.03% antioxidant reached the index of spoiled meat at the 3rd d. Therefore, the addition of 0.03% rosemary antioxidant could significantly inhibit microbial proliferation (P < 0.05), and the shelf-life of minced pork could be extended by 4 d compared with that of the control group.

 

3 Conclusion

Therefore, in the present study, a water-dispersible rosemary antioxidant was prepared from the fat-soluble extract of rosemary by emulsification and homogenization, and the optimum process conditions were determined by orthogonal experiments as follows: 1:5 (m/m) for PGFE and Tween 80, 4:6 (m/m) for oil-water phase, 2800 r/min emulsification, and homogenized at 20 MPa for 3 min to obtain the water-dispersible rosemary antioxidant, and the particle size was 318.83 nm. The water-dispersed rosemary antioxidant was obtained at 2800 r/min, and then homogenized at 20 MPa for 3 min to obtain the water-dispersed rosemary antioxidant, and the particle size was measured to be 318.83 nm. The water-dispersed rosemary antioxidant obtained under this condition had a small particle size. The application of the antioxidant to minced pork could effectively slow down the fat oxidation of minced pork during the storage process, and the best antioxidant effect was observed in minced pork with the addition of 0.03% antioxidant. The 0.03% water-dispersible rosemary antioxidant could also effectively inhibit the growth of microorganisms in pork mince, slow down the degree of spoilage, and stabilize the color of pork mince; the shelf-life of pork mince with 0.03% rosemary could be extended for 4 d compared with that of control group. Therefore, adding the fat-soluble extract of rosemary into pork mince as water-dispersible form could effectively slow down the oxidation of fat, improve the stability of pork mince and inhibit microorganism growth, thus prolonging the storage period of pork mince. It can effectively slow down the fat oxidation, improve the stability of minced pork and inhibit the growth of microorganisms, thus prolonging its shelf-life.

 

In this study, the antioxidant of rosemary can not only play the role of antioxidant, but also has color protection and bacteriostatic effects, showing multi-functions, and can be applied to meat products, or can reduce the dose of color protection and preservative, saving costs and improving consumer acceptance. However, the special flavor of rosemary extract may interfere with its flavor in some specific meat products, which is a problem that needs to be solved for the wide application of rosemary.

 

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