Abstract:
A method for preparing a shelf stable beverage is provided that includes blending ε-poly-L-lysine with a beverage and then pasteurizing at lower temperatures without a need of ultra high temperature treatment. The method is effective for providing a shelf stable beverage with improved microbial stability and better color and flavor quality than beverages processed at ultra high temperatures normally required for shelf stability.

Description:
[0001]     The invention is directed to a method for making shelf stable beverages which is effective for preventing growth of microbiological contaminants in beverages with reduced heat treatment. More specifically, the method includes adding low levels of ε-poly-L-lysine to beverages which is effective for making shelf stable products without employing ultra high temperature treatment normally required for microbial stability. The beverages containing the low levels of ε-poly-L-lysine may be subsequently processed at lower temperatures than ultra high temperatures without spoilage concerns. The low processing temperature treatment in the presence of low levels of ε-polylysine results in beverages having improved microbial stability, better color and flavor quality than beverages processed at the ultra high temperatures normally used due to spoilage concerns.  
       BACKGROUND  
       [0002]     Present food technologists utilize an array of physical, chemical, and biological processes and agents to preserve food. Many chemical compositions exist which kill or inhibit deleterious bacteria and/or other microbes thereby preserving food and beverages and preventing spoilage.  
         [0003]     Food preservation by inhibition of growth of microbiological contaminants is often difficult and is often done with the use of chemical compositions. A chemical composition added to foods should be effective for preventing increases in microbial numbers and should not add undesirable flavors or undesirable organoleptic properties to the food.  
         [0004]     The antimicrobial effect of ε-poly-L-lysine is well-known. More specifically, ε-poly-L-lysine is known to have some effect in delaying the growth of certain bacteria and fungi to some extent under certain conditions. For example, ε-poly-L-lysine has been used for making antimicrobial fabrics which can be used as food wrapper or container by kneading it together with the fabrics or directly spraying it on the fabrics (U.S. Pat. No. 5,759,844). Methods of producing ε-poly-L-lysine and its use are described in U.S. Pat. No. 6,294,183 (“Antimicrobial Resin Composition and Antimicrobial Resin Molded Article Comprising Same”); U.S. Pat. No. 5,294,552 (“Strain mass-producing ε-poly-L-lysine”); U.S. Pat. No. 5,434,060 (“Method for Producing ε-poly-L-lysine”); U.S. Pat. No. 5,759,844 (“Antibacterial Articles and Methods of Producing the Articles”); U.S. Pat. No. 5,900,363 (“Process for producing ε-poly-L-lysine with immobilized  Streptomyces albulus ”); U.S. Pat. No. 5,453,420 (“Food preservative and production thereof”); U.S. Pat. No. 5,009,907 (“Method for Treating Food to Control the Growth of Yeasts”); U.S. Pat. No. 4,597,972 (“Nisin as an Antibotulinal Agent for Food Products”); and U.S. Pat. No. 4,584,199 (“Antibotulinal Agents for High Moisture Process Cheese Products”). These patents are incorporated herein by reference in their entireties.  
         [0005]     The acid resistant and heat resistant bacteria such as spore-former  Alicyclobacillus acidoterrestris  has been a common spoilage organism for fruit-juice-containing ready-to-drink beverages. Controlling the growth of  A. acidoterrestris  in such beverages continues to be a big challenge in the beverage industry today. Production of a shelf-stable fruit juice-containing beverage generally requires ultra high temperature (UHT) to inactivate spores. Such temperature will not only increase the capital and operating costs, but also cause flavor problems in the beverages. A need exists for compositions which can be added to beverages which are effective for preserving the beverage and preventing spoilage without the need of UHT treatment while not adversely affecting the taste and physical properties of the beverage.  
       SUMMARY  
       [0006]     Ready-to-drink (RTD) beverages are formulated with an amount of ε-poly-L-lysine effective for improving microbial stability and flavor quality. By formulating a natural fermentation product, ε-poly-lysine, into RTD beverages, the growth of spoilage organisms such as thermophilic, acid-resistant and spore-former bacteria  Alicyclobacillus acidoterrestris  is effectively eliminated or inhibited in the beverage. The beverage may be subsequently processed with regular pasteurization temperatures instead of ultra high temperature treatment. Lower temperature processing significantly improves the color and flavor qualities of the beverage due to higher retention of natural flavor and color, minimized decomposition of the thermal liable flavor compounds and reduced formation of undesirable flavor in the beverage.  
         [0007]     The method as claimed is effective for preventing a reduction in volatile flavor compounds as compared to levels of volatile flavor compounds in beverages that did not contain ε-poly-L-lysine and which were heated to a temperature of greater than 240° F. In this aspect, the method is effective for reducing volatile flavor compounds in RTD beverages less than about 10%, in an important aspect, less than about 5%, and in another aspect, about 0%, as compared to RTD beverages heated at temperatures greater than about 240° F. without ε-poly-L-lysine.  
         [0008]     In another aspect, the method is effective for preventing the formation of off-flavor compounds. In this aspect, the method is effective for reducing formation of off-flavor compounds by at least about 40%, in an important aspect, about 50% and in a very important aspect about 60%, and in another important aspect greater than about 90%, as compared to RTD beverages heated at temperatures greater than about 240° F. without ε-poly-L-lysine.  
         [0009]     In another aspect, the method is effective for enhancing color retention in RTD beverages. In this aspect, the color of RTD beverages processed in accordance the present invention was similar to the color of unprocessed RTD beverages.  
         [0010]     RTD beverages are blended with about 5 ppm to about 100 ppm ε-poly-L-lysine, preferably about 10 ppm to about 50 ppm ε-poly-L-lysine. The RTD beverage is then heated to a temperature of about 200° F. to about 210° F. The inclusion of ε-poly-L-lysine allows for the lower pasteurization temperatures of about 200° F. to about 210° F. The combination of ε-poly-L-lysine and lower pasteurization temperature is effective for making a shelf stable beverage and/or eliminating spoilage microorganisms. Such combination is effective for reducing and/or preventing an increase in microbial counts in beverages of about 4 log or less after about 16 days, in an important aspect about 2 log or less after about 10 days, in another aspect about 1 log or less after about 10 days, all at a storage temperature of about 113° F. 
     
    
     DETAILED DESCRIPTION  
       [0011]     Formulating ε-poly-lysine into RTD beverages at relatively low concentrations inhibits spore regrowth such as the growth of spores of  Alicyclobacillus acidoterrestris  in the beverages, without the need for severe heat treatment to make a shelf stable beverage. This provides significant cost advantage and quality improvement for the beverages, as the processing temperature can be changed from UHT to regular pasteurization temperature. The color and flavor quality of the RTD beverages formulated with ε-poly-lysine was significantly improved.  
         [0000]     Definitions  
         [0012]     “Food preservation”, as that term is used herein, includes methods which delay or prevent food spoilage due to microbes. Food preservation keeps food safe for consumption and inhibits or prevents nutrient deterioration or organoleptic changes causing food to become less palatable.  
         [0013]     “Food spoilage”, as that term is used herein, includes any alteration in the condition of food which makes it less palatable including changes in taste, smell, texture or appearance. “Ultra High Temperatures” or “UHT” refers to processing at temperatures of 240° F. to about 250° F. “Pasteurization” refers to about 200° F. to about 210° F.  
         [0014]     As used herein, “shelf stable beverage” or “shelf stable beverages” means that the beverage has no increase in microbial growth after storage at room temperature (25° C.) for at least about 4 weeks, preferably at least about 8 weeks.  
         [0015]     As used herein, “eliminating spoilage microorganisms” means that less than 1 cfu/ml of spoilage organisms are detected in a beverage after storage at room temperature (25° C.) for at least about 6 days, preferably at least about 10 days, and more preferably at least about 16 days.  
         [0016]     As used herein, “volatile flavor compounds” refers to compounds such as ethyl acetate, ethyl butyrate, ethyl 2-methyl butyrate, 2-methyl butyl acetate, beta-pinene, butyl-2-methyl isobutyrate, ethyl caproate, hexyl acetate, octanal, leaf alcohol, nonanal, trans-2-hexenol, ethyl caprylate, p-dimethyl styrene, heptanol, furfural, 2-ethyl hexanol, decanal, benzaldehyde, 4-terpineol, dihydrovarvone, beta-terpinol, alpha-terpineol, citral (neral, geranial), beta-citronellol, damacenone, carveol, geraniol, caprylic acid and gamma-decalactone. These compounds alone or in combination provide desirable flavor components to the RTD beverage.  
         [0017]     “Off-flavor compounds” refers to compounds formed during heat treatment such as sugar degradation products. “Sugar degradation products” refers to undesirable volatile compounds which form with heat treatment of sugars. An example of a sugar degradation products includes furfanol. An example of an off flavor compound includes para-dimethyl styrene.  
         [0000]     Ready-to-Drink Beverages  
         [0018]     Ready-to-drink (RTD) beverages may include high acid, juice containing beverages such as fruit punch, and 100% fruit juice beverages such as apple juice, as well as high acid, non-juice containing beverages such as sport drinks.  
         [0000]     ε-poly-L-lysine  
         [0019]     ε-poly-lysine is a polymer of L-lysine produced by  Streptomyces albulus  through natural fermentation, and it has limited applications as a preservative in several ready-to-eat food products such as cooked rice and sushi for shelf life extension. Since it is fungistatic and bacteriostatic, it is generally intended for limited shelf life extension of selected food products.  
         [0020]     ε-poly-L-lysine can be used as a free type or a salt type of an inorganic acid such as hydrochloric acid, sulfuric acid or phosphoric acid, or an organic acid such as acetic acid, propionic acid, fumaric acid, malic acid or citric acid. Both types of these salts of inorganic acids and organic acids as well as a free type have similar antibacterial effect. The ε-poly-L-lysine has the structure  
                         
 
 where n is about 25 to about 35. 
 
         [0021]     ε-Poly-L-lysine is available under the tradename Save-ory™ GK128 from Chisso Corporation in Japan. This commercial preparation contains 1.0% ε-poly-L-lysine as the active antimicrobial agent, 30% glycerin, 68.8% water, a trace amount of organic acids for pH adjustment, and emulsifier. Save-ory™ products have been used in sushi and cooked rice for shelf-life extension in Japan.  
       EXAMPLES  
     Example 1  
     Preparation of  Alicyclobacillus acidoterrestris  Spores  
       [0022]     A strain of  A. acidoterrestris  isolated from a fruit juice-containing beverage was incubated on acidified potato dextrose agar (PDA, pH adjusted to 3.5 with tartaric acid) at 45° C. for 5-7 days. The resulting cells and spores on the PDA plate were then harvested by washing with 10 ml of sterile water. The suspension was centrifuged at 5,000 rpm for 10 min. The supernatant was poured off and the precipitate was re-suspended in 10 ml of sterile water and the washing cycle was repeated. After 2-3 washing cycles, the cell and spore suspension was heated in a water bath at 80° C. for 10 min to eliminate vegetative cells. After heat shock, the spore suspension was cooled in an ice/water bath to ambient temperature, and was serially diluted to the desired concentrations for inoculation studies. The spore concentration was determined by plate count on the acidified PDA plate incubated aerobically at 45° C. for 2-3 days.  
       Example 2  
     Inhibition of  A. acidoterrestris  in a Fruit Juice-Containing Beverage at High Inoculation Level  
       [0023]     A beverage was provided that contained 10% mixed fruit juices with a pH of 3.5 and no preservatives. The  A. acidoterrestris  spores prepared in Example 1 were inoculated into the beverage at an inoculation level of ˜1×14 spores/ml. The beverage samples contained various levels of ε-poly-L-lysine as indicated in Table 1. The ε-poly-L-lysine used in the experiment was obtained from Chisso Corporation as a liquid product Save-ory GK128 that contains 1% ε-poly-L-lysine. After about two weeks of incubation at 45° C. the control had  A. acidoterrestris  grown to 8×10 4  cfu/ml, while the samples containing 20 and 50 ppm ε-poly-L-lysine (0.2% and 0.5% GK128, respectively) had 3-4 log lower cell count compared to control. The sample with 50 ppm ε-polylysine also had 3 log reduction in 10 days compared to the initial inoculation level.  
                                         TABLE 1                           Inhibition of  A. acidoterrestris  in a fruit juice-containing       beverage at high inoculation levels            Treatment   Day 0   Day 2   Day 6   Day 10   Day 16               Control   8.3 × 10 3     9.7 × 10 3     1.8 × 10 5     1.0 × 10 5     8.0 × 10 4         0 ppm       polylysine       5 ppm   8.1 × 10 3     9.5 × 10 3     1.6 × 10 5     1.1 × 10 5     3.1 × 10 4         polylysine       10 ppm   6.4 × 10 3     7.4 × 10 3     2.7 × 10 5     8.9 × 10 4     1.2 × 10 4         polylysine       20 ppm   6.2 × 10 3     6.8 × 10 1     1.7 × 10 2     1.6 × 10 1     1.4 × 10 1         polylysine       50 ppm   7.7 × 10 3     4.0 × 10 1     1.6 × 10 2     8   7       polylysine                 Note:            All numbers are in cfu/ml.             
 
       Example 3  
     Inhibition of  A. acidoterrestris  in a Fruit Juice-Containing Beverage at Low Inoculation Levels  
       [0024]     The  A. acidoterrestris  spores prepared in Example 1 were inoculated into the fruit juice-containing beverage described in Example 2 at a target level of 10 spores/ml. The beverage samples contained various concentrations of GK128 as described in Example 2. After incubation at 45° C. for about 2 weeks, the control had  A. acidoterrestris  grown to 8×10 4  cfu/ml while the samples containing 20 and 50 ppm ε-poly-L-lysine (0.2% and 0.5% GK128, respectively) did not have live cells detected (Table 2).  
                                         TABLE 2                           Inhibition of  A. acidoterrestris  in a fruit juice-containing       beverage at lower inoculation levels            Treatment   Day 0   Day 2   Day 6   Day 10   Day 16               Control   2.0 × 10 1     6.6 × 10 2     1.9 × 10 5     1.1 × 10 5     8.0 × 10 4         0 ppm       polylysine       5 ppm   2.0 × 10 1     5.0 × 10 3     6.1 × 10 4     5.8 × 10 4     4.9 × 10 4         polylysine       10 ppm   2.0 × 10 1     2.0 × 10 3     1.3 × 10 4     6.3 × 10 3     6.2 × 10 3         polylysine       20 ppm   2.0 × 10 1     &lt;1   &lt;1   &lt;1   &lt;1       polylysine       50 ppm   2.0 × 10 1     &lt;1   &lt;1   &lt;1   &lt;1       polylysine                 Note:            All numbers are in cfu/ml             
 
       Example 4  
     Inhibition of  A. acidoterrestris  in a Non Juice-Containing Beverage  
       [0025]     A beverage was provided that did not contain fruit juice but contained fruit flavor, had a pH of 3.5 and did not contain any preservatives. The beverage was made of water, high fructose corn syrup, sugar, citric acid, sodium citrate and potassium citrate and small amount of natural fruit flavor. The  A. acidoterrestris  spores made in Example 1 were inoculated at the target level of 10 4  spores/ml into the beverage which contained ε-poly-lysine at the concentration levels ranging between 5 and 50 ppm. After the samples were incubated at 45° C. for about 2 weeks, the control had  A. acidoterrestris  at the 10 4  cfu/ml while all the polylysine-containing samples had less than 10 cfu/ml (Table 3).  
                                         TABLE 3                           Inhibition of  A. acidoterrestris  in a non juice-containing beverage            Treatment   Day 0   Day 2   Day 6   Day 10   Day 16               Control   7.2 × 10 3     2.1 × 10 2     1.3 × 10 5     8.3 × 10 4     2.6 × 10 4         0 ppm       polylysine       5 ppm   7.9 × 10 3     3.1 × 10 2     3.0 × 10 1     2.1 × 10 1     1       polylysine       10 ppm   7.9 × 10 3     5.5 × 10 2     7.0 × 10 1     3.8 × 10 1     4       polylysine       20 ppm   8.0 × 10 3     2.9 × 10 2     1.2 × 10 2     5.9 × 10 1     3       polylysine       50 ppm   6.8 × 10 3     4.9 × 10 2     1.4 × 10 2     6.7 × 10 1     6       polylysine                 All numbers are in cfu/ml             
 
       Example 5  
     Microbial Stability of a Fruit Juice-Containing Beverage Prepared with Reduced Heat Treatment  
       [0026]     The current fruit juice-containing beverage as described in Example 2 is produced with ultra high temperature treatment (UHT, 243° F. for 1.3-6 seconds). To test the feasibility of reducing the process temperature with the use of ε-polylysine, a pilot plant trial was run with the following variables: 1) Control (no polylysine, heat treatment at 243° F., hot fill), 2) Reduced heat treatment (30 ppm polylysine or 0.3% GK128, heat treatment at 205° F., cold fill). After storage for 4 and 8 weeks, total microbial count was conducted in both samples. The results suggest that the reduced heat-treated sample was as microbial stable as the UHT-treated control (Table 4).  
                                 TABLE 4                           Microbial stability of a juice-containing beverage prepared       with reduced heat treatment and UHT treatment                Treatment   4 weeks   8 weeks                       No polylysine, heat 243° F., hot fill   No growth   No growth           30 ppm polylysine, heat 205° F., cold   No growth   No growth           fill                      
 
       Example 6  
     Color Improvement of a Fruit Juice-Containing Beverage with Added Polylysine and Reduced Heat  
       [0027]     The fruit juice-containing beverages were prepared as in the Example 5. After storage at ambient temperature for 8 weeks, the color of the treatment sample was significantly superior to the control.  
         [0028]     The color of these samples was evaluated in comparison with the original unprocessed juice beverage by Hunter Lab color measurement and their respective L, a, b values are listed in Table 5. The L value represents transparency of the sample. The positive a value means red and negative value is green. The positive b value is yellow and negative value means blue. The data in Table 5 suggest that the low heat treatment, cold filled, polylysine-containing sample (C) had more intense red color and less yellow color than the regular hot filled product (B). The differences in these values compared to the unprocessed sample (A) were expressed as delta values (ΔL, Δa and Δb). These values indicate how close the processed products were to the unprocessed sample in color. The data show that sample C was less transparent but its color was closer to sample A than sample B (Table 5). These results suggest that the polylysine treated sample had a better color retention than the currently UHT processed product.  
                                                                   TABLE 5                           Hunter Lab color measurement of fruit       juice-containing beverage samples            Sample   L value (ΔL)   a value (Δa)   b value (Δb                    A.   Unprocessed,   10.48   (0)   0.76   (0)   −1.23   (0)           no heat       B.   No polylysine,   9.11   (−1.37)   0.35   (−0.41)   0.15   (+1.38)           heat 243° F.,           hot fill       C.   30 ppm   7.24   (−3.24)   0.78   (+0.02)   0.06   (+1.29)           polylysine,           heat 205° F.,           cold fill                  
 
       Example 7  
     Flavor Improvement of a Fruit Juice-Containing Beverage with Added Polylysine and Reduced Heat  
       [0029]     Three fruit juice-containing beverage samples were prepared as follows. Sample A (control), No polylysine, heat treated to 243° F., hot filled; Sample B, 30 ppm polylysine (0.3% GK128), heated to 243° F., hot filled; Sample C, 30 ppm polylysine, heated to 205° F., cold filled. After initial storage at ambient temperature for 4 weeks, all samples were tasted by a trained sensory panel to compare the flavor quality. The sensory evaluation results suggest that the polylysine-containing sample with reduced heat treatment had superior appearance, color and taste over the control sample. To further quantify the flavor benefits of the treatment, a comprehensive flavor characterization test was conducted after 8 weeks of storage.  
         [0030]     Each sample was freeze concentrated to increase the flavor concentration, and the flavor isolation was done by Solid Phase Micro Extraction (SPME) at 40° C. for 30 minutes. The flavor components were identified by GC-MS and were quantified by GC-FID. The flavor compounds identified for flavor stability evaluation in the beverage included ethyl acetate, ethyl butyrate, ethyl 2-methyl butyrate, 2-methyl butyl acetate, beta-pinene, butyl-2-methyl isobutyrate, ethyl caproate, hexyl acetate, octanal, leaf alcohol, nonanal, trans-2-hexenol, ethyl caprylate, p-dimethyl styrene, heptanol, furfural, 2-ethyl hexanol, decanal, benzaldehyde, 4-terpineol, dihydrovarvone, beta-terpinol, alpha-terpineol, citral (neral, geranial), beta-citronellol, damacenone, carveol, geraniol, caprylic acid and gamma-decalactone.  
         [0031]     The loss of volatile flavor compounds in the beverage samples was determined by comparing their relative concentrations. The concentration of each compound in the polylysine-treated sample was set to 100% and the UHT-treated control sample was compared to the treatment sample to obtain relative concentration of the flavor compound. The relative concentrations of some key volatile flavor compounds (esters) in the polylysine treated sample and the control is shown in Table 6.  
                                                                       TABLE 6                           Relative concentrations of volatile compounds       in fruit juice-containing beverage samples                        Ethyl 2-                   Ethyl   Ethyl   methyl   Ethyl   Ethyl       Sample   acetate   butyrate   butyrate   caproate   caprylate                    A.   No polylysine,   49   74   77   75   78           243° F., hot fill       B.   Polylysine,   56   76   80   86   89           243° F., hot fill       C.   Polylysine,   100   100   100   100   100           205° F., cold fill                  
 
         [0032]     Clearly sample A had much less retention of volatile compounds than sample C (Table 6). These volatile compounds are important components of the fruit juice-containing beverages. The polylysine treatment with reduced heat process significantly increased the retention of volatile flavor compounds, and therefore improved the flavor quality of the beverage.  
         [0033]     Some known heat-sensitive flavor compounds such as neral, citronellol and geranial were degraded during stack burn of hot filled beverages. The relative concentrations of these compounds in the samples were listed in Table 7. The UHT-treated sample (A) lost about 80-90% of these flavor compounds as compared to the polylysine-containing sample with reduced heat treatment (C). Neral and geranial are key citral aroma compounds and they are responsible for the fresh lemon smell. Formulating polylysine in the beverage could reduce the heat treatment and therefore improve the flavor quality of the product.  
                                                                   TABLE 7                           Relative concentrations of thermal liable flavor       compounds in fruit juice-containing beverage                Sample       Neral   Citronellol   Geranial                            A.   No polylysine,   11   23   11               243° F., hot fill           C.   Polylysine,   100   100   100               205° F., cold fill                      
 
         [0034]     The heat treatment also resulted in sugar degradation. The sugar degradation produced undesirable volatile compounds such as furfural. The concentration of furfural in the samples was also measured. The UHT-treated product produced more than two times the amount of furfural than the product with reduced heat treatment (Table 8). Higher concentrations of furfural could contribute jammy-like and burnt sugar-like flavor. By formulating polylysine the product could be processed at lower temperature. This significantly reduced the formation of undesirable flavor compounds such as furfural, and therefore improved the flavor quality of the beverage.  
                             TABLE 8                           Relative concentrations of heat-induced undesirable       volatile compound in fruit juice-containing beverage                Sample   Furfural                       A. No polylysine, 243° F., hot fill   234           C. Polylysine, 205° F., cold fill   100                      
 
         [0035]     In addition, the product with reduced heat treatment also minimized the formation of other off-flavor compounds. For example, an off-flavor compound para-dimethyl styrene was detected in the UHT-treated and hot filled product but not detected in the polylysine-containing sample with reduced heat-treatment. The para-dimethyl styrene is known degradation product of citral under high temperature in a low pH environment. It has a gasoline-like or turpentine off-flavor.