Abstract:
Improved methods are provided for the treatment of foods to inhibit microbial growth therein, by the applying to or incorporating into the food a quantity of an alkali or alkaline earth metal salt of levulinic acid. The salts may be topically applied by spraying or dipping, or can be incorporated into the foods such as comminuted meat products. Preferred antimicrobial agents are the sodium and potassium salts of levulinic acid, applied or incorporated at a level of from about 0.5-4% by weight. A variety of foods may be treated in accordance with the invention, such as human foods (e.g., meats and cheeses) or animal foods (pet foods).

Description:
BACKGROUND OF THE INVENTION 
     1. Field of the Invention 
     The present invention is broadly concerned with improved methods for the treatment of foods such as meats and cheeses to inhibit microbial growth, as well as the food products themselves. More particularly, the invention is concerned with treatment of foods with a minor amount of an alkali or alkaline earth metal salt in liquid or solid form for antimicrobial purposes. 
     2. Description of the Prior Art 
     The growth and proliferation of many types of microbes can materially affect the quality of food products. It has been known in the past to treat meat products with steam, hot water wash, acid wash and addition of antimicrobial substances such as sodium lactate, sodium acetate, sodium diacetate, and nisin. Lactates are commonly added to processed meats because their antimicrobial activity increases shelf-life. Lactates used at a level of 1.5-3% of meat weight has been used extensively in the industry as an antimicrobial additive and to improve various quality attributes of meat. Lactates tend to positively affect flavor, microbial shelf-life and safety of pork. 
     Levulinic acid (4-oxopentanoic acid, C 5 H 8 O 3 ) is a 5-carbon organic acid commercially available as a biproduct of corn extrusion. Levulinic acid is known to have prooxidative activity that could limit shelf-life (Yi et al. “Antioxidant Activity of Maltol, Kojack Acid, Levulinic Acid, Furfural, 5-hydroxymethyl furfural, and Pyrazine.”  Kor. J. Food Sci.  14, 265-270 (1982)). 
     Various methods have been devised in the past for the production of levulinic acid. For example, U.S. Pat. No. 5,859,263 describes an efficient method for the production of levulinic acid via reactive extrusion. 
     Conversion of levulinic acid to the corresponding salts is generally quite straight forward, involving neutralization with appropriate cations and proper maintenance of pH to assure that the salt form predominates. 
     SUMMARY OF THE INVENTION 
     The present invention is directed to new methods for treatment of foods to inhibit microbial growth by applying to or incorporating into food a quantity of an alkali or alkaline metal salt of levulinic acid. A wide variety of foods (for human or animal, e.g., pets, consumption) may be treated in accordance with the invention, but the most preferred foods are meats (either comminuted or whole muscle) and cheeses. The levulinic salt may be applied by spraying or dipping in the case of whole muscle meats or cheeses, when use is made of a liquid salt dispersion or solution. Alternately, and especially in the case of comminuted food products, the salt in dried form may be incorporated into the foods by thorough mixing. In such cases the native moisture of the food serves to ensure dissilution and even application of the salt. 
     The levulinic salts of the invention may be applied or incorporated at a level of from about 0.5-4% by weight, based upon the weight of the food taken as 100% by weight. More preferably, the level of use if from about 1-3% by weight, particularly in the case of meat products. Various salts may be used, but for reasons of cost and availability the sodium, potassium and calcium salts of levulinic acid are preferred. 
     The levulinic salt-supplemented food products of the invention exhibit significantly enhanced microbial properties, compared with untreated foods or those treated with conventional antimicrobial agents such as sodium lactate. In actual tested food systems, levulinic acid was found not to exhibit prooxidant activity, since TBA values were low for such samples. 
    
    
     
       BRIEF DESCRIPTION OF THE DRAWINGS 
         FIG. 1  is a comparative graph of aerobic plate count versus time for fresh pork sausage samples treated with sodium levulinate and sodium lactate, respectively; and 
         FIG. 2  is a comparative graph of aerobic plate count versus time for fresh turkey sausage samples treated with sodium levulinate and sodium lactate, respectively. 
     
    
    
     DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENT 
     The following example sets forth a study of the comparative antimicrobial properties of sodium levulinate versus sodium lactate with fresh pork and turkey sausages. 
     MATERIALS AND METHODS 
     Apparatus 
     The equipment used in this study included: Hobart grinder model 4152 (Hobart Mfg. Co., Troy, Ohio); Miniscan XE Plus portable colorimeter Model 45/O-S (Hunter Associates Laboratory Inc., Reston, Va.); Pressure cooker (16 L capacity; Mirromatic, Mirro Corp., Manitowoc, Wis.); pH meter (Fisher Accumet, model 610A, Houston, Tex.), pH meter electrode (Coming G-P combo electrode, Corning, N.Y.); Wrapping and sealing equipment (Heat Sealing Manufacturing Co., Cleveland, Ohio). 
     Experimental Design and Statistics 
     Water or syrups (15% and 30% w/w) of sodium lactate and sodium levulinate were added to fresh pork sausage at 10% of the meat weight. The sausage mix was divided into 5 portions, then placed on styrofoam trays, over-wrapped with PVC film and stored at 2 C. A single package from each treatment was removed at days 0, 1, 3, 7 and 14 and color, aerobic plate count, pH and TBA values were monitored, and fat content was measured on the 0 day samples. The experiment was replicated 3 times for both pork and turkey sausage. 
     The data were analyzed using STATISTICA (Statsoft Inc., Tulsa, Okla.) software. The effect of replication was blocked in order to avoid the variations due to the different meat batches used for each replicate. 
     Data were analyzed by MANOVA as a complete factorial (2*2+1) in a split plot model. The whole plot factors were salt type (levulinic or lactic), level of salt in sausage (1.4 or 2.7%), or water control. Whole plot factors had n=3 reflecting independent observations from three separate batches of sausage. Storage time in days was the subplot factor. To compare means, LSD value was calculated when P&lt;0.05. 
     Preparation of Sodium Lactate and Sodium Levulinate 
     A 30% sodium lactate solution (pH 5.9-6.0) was prepared using a 60% sodium lactate solution (Sigma Chemicals, DL-Lactic acid sodium salt, 60% (w/w) syrup) and diluting it 1:1 with distilled water. Sodium levulinate solution was prepared by adding 20% NaOH to a 98+% levulinic acid solution (Sigma Chemicals, St. Louis, Mo.) to obtain a pH of 6.59. Because the pKa of levulinic acid is 4.59, the 2-pH unit difference due to titration with NaOH ensures that 99% of the levulinic acid was in the form of its sodium salt. The resulting levulinate was diluted with sufficient distilled water to produce a 30% solution with a pH of 6.6-6.7. The 30% solutions of sodium lactate and sodium levulinate were diluted 1:1 with distilled water to produce 15% solutions. These solutions were then added to the meat samples at the rate to 10% w/w of meat weight. 
     Sample Preparations 
     Approximately 6 kg of fresh pork lean trim or 6 kg fresh turkey breast meat were coarsely ground once through a grinder plate with 0.64 cm diameter pore size (Hobart Mfg. Co., Troy, Ohio). Premixed spices (Heller JD&#39;s country pork sausage seasoning GD-5076, Modesto, Calif.) were added to the sausage at the rate of 0.02 kg per kg meat and the meat mixture was passaged again through the coarse grinder plate. After adding the premixed spices, sausages contained 1.2% salt (no phosphate). The mixture was divided into 5 portions of 1 kg and each portion was mixed with either 10% water (control), 15% lactate, 30% lactate, 15% levulinate or 30% levulinate solutions. The coarsely ground mixtures were passed through the fine grinder plate (0.32 cm diameter pore size). Each mixture was further divided into 5 samples of 200 g each, packaged on a styrofoam tray and tightly over-wrapped with an oxygen-permeable polyvinyl chloride film (SWM select wrap, Anchor Packaging, Marmaduke, Ark.) and stored in the USU meat laboratory product cooler for 0, 1, 3, 7 and 14 days at 2 C. 
     Color Measurement 
     Color was measured on day 0, 1, 3, 7 and 14 using Miniscan XE Plus portable colorimeter Model 45/O-S (Hunter Associates Laboratory, Reston, Va.). The illuminant setting was D65, with 10° standard observer angle. The instrument was standardized using a white and black standard plate. The sample L8, a* and b* values were then determined, and hue angle=tan −1 (b*/a*) was calculated. Larger hug-angle values are associated with less red color (Van Laack et al. 1996), where hue-angle 0=red and hue-angle 90=yellow. The saturation index, corresponding to color intensity was also calculated (a *2 +b *2 ) 1/2 . 
     Aerobic Plate Count 
     Aerobic plate counts of meat samples were determined by placing 10 g samples into 90 mL of 0.1% sterile bacto-peptone solution (Difco, Detroit, Mich.). Serial dilutions were pour plated following standard procedures (Messer et al. 1978). Standard plate count agar (Difco, Detroit, Mich.) was used as growth media. Pork samples were incubated at 37 C. for 48 h. Turkey samples were incubated for 60 h at 37 C. to obtain larger colonies that could be distinguished from meat particles at 10 −1  dilution. 
     TBA Values 
     Thiobarbituric acid-reactive substances (TBARS) were determined as described by Buege and Aust (1978). Duplicate samples (0.5 g) for each treatment were mixed with 2.5 mL stock solution containing 0.375% TBA (Sigma Chemical Co., St. Louis, Mo.), 15% trichloroacetic acid (Mallinckrodt Baker, Paris, Ky.), and 0.25 N HCl. Samples were then heated for 10 min in a boiling water bath (100 C) to develop pink color, cooled under tap water, and centrifuged (Sorvall Instruments, Model RC 5C, Dupont, Wilmington, Del.) at 12,465×g for 10 min (rather than 1000×g as specified by Beuge and Aust 1978), to reduce sample turbidity. Absorbance was determined at 532 nm (Spectronic 21D, Milton Roy, Rochester, N.Y.) against a blank containing all reagents other than meat. The malonaldehyde (MDA) concentration was calculated using an extinction coefficient of 1.56×10 5−1  cm −1  (Sinnhuber and Yu 1958). MDA concentration was converted to TBA number (mg MDA/kg meat sample) as follows:
         (1) TBA number (mg/kg)=sample A 532 ×(1M MDA/1.56×10 5 )×[(1 mole/L)/M]×(0.003 L/0.5 g meat)×(72.07 g MDA/mole MDA)×(1000 mg/g)×(1000 g/kg) or   (2) TBA number (ppm)=sample A 532 ×2.77
 
pH Values
       

     The pH values for each sample were determined by adding 90 mL deionized water to 10 g sample. The samples were then mixed with a glass rod and filtered through Whatman filter paper No. 2 (Fisher Scientific, Salt Lake city, Utah). The pH of the filtrate was determined using a pH meter calibrated at pH 4.0 and 7.0 (Fisher Chemicals, Fair Lawn, N.J.). 
     Fat Content 
     The fat content of the samples was determined using AOAC procedure 960.39 for solvent extraction of crude fat (AOAC 1990). 
     RESULTS 
     Microbial load of both pork and turkey sausages were significantly (P&lt;0.05) affected by treatments, storage time and the interaction of treatment and storage time. By day 14 of storage of pork sausages, both control and sausages with 1.4% lactate had a high log 10 APC (6.23 and 5.90, respectively,  FIG. 1 ). In comparison, pork sausage containing 2.7% sodium lactate, and both 1.4% and 2.7% sodium levulinate had significantly lower bacterial loads after 14 days storage with log 10 APC of 4.78, 4.42 and 4.29, respectively (LSD 0.05 =0.59,  FIG. 1 ). 1.4% sodium levulinate was a more effective antimicrobial agent than 1.4% sodium lactate in fresh pork sausage after 14 days storage with log 10 APC of 4.42 and 5.90, respectively (LSD 0.05 =0.59,  FIG. 1 ). 
     After 14 days of storage, the control turkey sausages had a mean log 10 APC of 5.45, which was significantly higher than all other treatments ( FIG. 2 ). Turkey sausage containing 2.7% lactate, and both levels of levulinate, had lower APC after 14 days than those containing 1.4% lactate ( FIG. 2 ). 1.4% sodium levulinate was as effective as 2.7% sodium levulinate for inhibition of aerobic microbial growth, as was the case for pork sausage. 
     TBA number of uncooked pork or turkey sausages was not affected by addition of sodium lactate or sodium levulinate. All TBA values for pork sausages were low ranging from 0.20 to 0.32 (Table 2). Most turkey sausage samples also had TBA values less than 0.5 (Table 2). TBA numbers of uncooked pork and turkey sausages decreased slightly, but significantly, during storage (Tables 1 and 2). 
     Storage time significantly affected Hunter color redness (a*), hue angle and saturation index of pork sausages (Table 1). Redness (a*) values decreased with storage time from 6.1 in day 0 controls to 4.9 after 14 days storage (Table 2). Hue angle of pork sausages increased form 65.8 to 69.7, indicating loss of redness during storage (Table 1). 
     Storage time significantly affected all Hunter color parameters of turkey sausages (Table 1). L*, a* and b* and saturation index values decreased with storage time, while the hue angle values increased indicating loss of redness during storage (Table 2). Lightness (L*) values were significantly affected by treatment (Table 1). Samples treated with 2.7% sodium levulinate had significantly higher L* values at day 0 than control treatment (L*=44.3 and 41.3, respectively; LSD 0.05 =2.38; data not shown in tabular form) 
     Sausage pH was slightly but significantly increased from 5.8 for controls to 5.9 with 2.7% levulinate in turkey sausages (data not shown). Both pork and turkey sausages were lean (13.6+0.9% and 3.6+0.6% fat, respectively). Sensory evaluation was not formally conducted. However, informal panels indicated no flavor changes associated with use of levulinate compared to controls. 
     
       
         
               
             
               
               
               
               
               
               
               
               
               
               
             
           
               
                 TABLE 1 
               
             
             
               
                   
               
               
                 SUMMARY OF SIGNIFICANCE (P &lt; 0.05) AS DETERMINED BY ANALYSIS 
               
               
                 OF VARIANCE (ANOVA) 
               
             
          
           
               
                 Meat 
                   
                   
                   
                   
                   
                   
                 Hue 
                 Saturation 
                   
               
               
                 Type 
                 Effect 
                 log 10  APC 1   
                 TBA 1   
                 L* 1   
                 a* 1   
                 b* 1   
                 angle 1   
                 index 1   
                 pH 
               
               
                   
               
               
                 Pork 
                 Treatment 2   
                 * 4   
                 NS 4   
                 NS 
                 NS 
                 NS 
                 NS 
                 NS 
                 NS 
               
               
                   
                 Storage time 3   
                 * 
                 * 
                 NS 
                 * 
                 NS 
                 * 
                 NS 
                 NS 
               
               
                   
                 Treatment * time 
                 * 
                 * 
                 NS 
                 NS 
                 NS 
                 NS 
                 NS 
                 NS 
               
               
                 Turkey 
                 Treatment 
                 * 
                 NS 
                 * 
                 NS 
                 NS 
                 NS 
                 NS 
                 * 
               
               
                   
                 Storage time 
                 * 
                 * 
                 * 
                 * 
                 * 
                 * 
                 * 
                 * 
               
               
                   
                 Treatment * time 
                 * 
                 NS 
                 NS 
                 NS 
                 NS 
                 NS 
                 NS 
                 NS 
               
               
                   
               
               
                   1 APC = log 10  aerobic plate count/g sample, TBA = Thiobarbituric acid number, L* = lightness; a* = redness; b* = yellowness; Hue angle = tan −1  (b*/a*), where lower values indicate more redness; Saturation index = (a* 2  + b* 2 ) 1/2   
               
               
                   2 Treatments were control (water), 1.5% or 3.0% sodium lactate or sodium levulinate 
               
               
                   3 Storage times were 0, 1, 3, 7 and 14 days at 2 C. 
               
               
                   4 * = significant at P &lt; 0.05; NS = not significant at P &lt; 0.05. 
               
             
          
         
       
     
     
       
         
               
             
               
               
               
               
               
               
               
               
             
           
               
                 TABLE 2 
               
             
             
               
                   
               
               
                 POOLED MEANS BY STORAGE TIME FOR TBA VALUES, pH, AND HUNTER COLOR 
               
               
                 PARAMETERS OF FRESH PORK AND TURKEY SAUSAGES (TREATMENT EFFECTS 
               
               
                 WERE POOLED) 
               
             
          
           
               
                 Meat type and 
                   
                   
                   
                   
                 Hue 
                 Saturation 
                   
               
               
                 storage time 
                 TBA 1   
                 L* 1   
                 a* 1   
                 b* 1   
                 angle 1   
                 index 1   
                 pH 
               
               
                   
               
               
                 Pork 
                   
                   
                   
                   
                   
                   
                   
               
               
                  0 (days) 
                     0.32 ± 0.14 2   
                 46.6 ± 8.2 
                 6.1 ± 0.6 
                 13.6 ± 2.0 
                 65.8 ± 2.2 
                 14.9 ± 2.0 
                 6.14 ± 0.15 
               
               
                  1 
                 0.32 ± 0.09 
                  47.7 ± 11.6 
                 6.6 ± 0.9 
                 14.0 ± 2.6 
                 64.6 ± 3.0 
                 15.5 ± 2.6 
                 6.14 ± 0.25 
               
               
                  3 
                 0.30 ± 0.11 
                 46.6 ± 8.0 
                 6.1 ± 0.5 
                 13.7 ± 1.5 
                 65.8 ± 2.8 
                 15.0 ± 1.3 
                 6.23 ± 0.10 
               
               
                  7 
                 0.24 ± 0.09 
                 48.3 ± 8.6 
                 5.6 ± 1.1 
                 13.5 ± 1.9 
                 67.5 ± 4.4 
                 14.6 ± 1.9 
                 6.19 ± 0.09 
               
               
                 14 
                 0.20 ± 0.09 
                 47.6 ± 8.6 
                 4.9 ± 1.3 
                 13.2 ± 2.3 
                 69.7 ± 4.7 
                 14.1 ± 2.4 
                 6.21 ± 0.10 
               
               
                 LSD 0.05   
                 0.07 
                 NS 
                 0.67 
                 NS 
                 2.58 
                 NS 
                 NS 
               
               
                 Turkey 
               
               
                  0 
                 0.52 ± 0.17 
                 42.8 ± 2.2 
                 2.2 ± 0.2 
                 10.6 ± 0.6 
                 78.1 ± 0.9 
                 10.8 ± 0.6 
                 5.87 ± 0.06 
               
               
                  1 
                 0.65 ± 0.18 
                 40.3 ± 1.5 
                 2.2 ± 0.2 
                 10.3 ± 0.7 
                 77.8 ± 0.8 
                 10.5 ± 0.7 
                 5.88 ± 0.05 
               
               
                  3 
                 0.52 ± 0.13 
                 39.4 ± 0.9 
                 2.0 ± 0.1 
                  9.9 ± 0.6 
                 78.8 ± 0.7 
                 10.1 ± 0.6 
                 5.80 ± 0.09 
               
               
                  7 
                 0.43 ± 0.09 
                 37.4 ± 1.4 
                 2.0 ± 0.1 
                  9.3 ± 0.8 
                 77.3 ± 4.3 
                  9.6 ± 0.8 
                 5.83 ± 0.05 
               
               
                 14 
                 0.49 ± 0.09 
                 36.9 ± 1.2 
                 1.5 ± 0.4 
                  8.8 ± 0.6 
                 80.4 ± 2.1 
                  9.0 ± 0.6 
                 5.84 ± 0.06 
               
               
                 LSD 0.05   
                 0.1  
                 1.12 
                 0.15 
                 0.50 
                 1.65 
                 0.50 
                 0.05 
               
               
                   
               
               
                   1 TBA = Thiobarbituric acid number, L* = Lightness; a* = redness; b* = yellowness; Hue angle = tan −1  (b*/a*), where lower values indicate more redness; Saturation index = (a* 2  + b* 2 ) 1/2   
               
               
                   2 Values were expressed as mean ± standard deviation for pooled treatment effects. 
               
               
                 NS = not significant at P &lt; 0.05. 
               
               
                 LSD 0.05  = Fisher&#39;s least significance difference at P &lt; 0.05. 
               
             
          
         
       
     
     This study demonstrated that 1.4% sodium levulinate was as effective an antimicrobial agent as 2.7% sodium lactate for both pork and turkey sausages. In both instances, no difference was found between 1.4% and 2.7% sodium levulinate on aerobic plate counts during storage. Thus, as to these products, there is no advantage by use of the higher level of sodium levulinate since the low level was quite effective. However, this was not the case with sodium lactate. The higher level (2.7%) of sodium lactate was more antimicrobial than the lower level (1.4%) for inhibition of bacterial growth in both pork and turkey sausages. This is in accordance with prior reports.