Abstract:
The invention provides process or method of malting cereal seeds, such as barley, wheat, rye, corn, oats, rice, millet and sorghum which process includes reducing the microbial level in the seeds prior to steeping, malting and the introduction of a starter culture used in the malting process According to the invention, the reduction of microbial levels permits efficient use of starter cultures, such as molds, yeasts, bacteria, spores and activated spores.

Description:
[0001]    This application is a continuation-in-part application of application Ser. No. 09/461,890, filed Dec. 15, 1999, which application is a continuation-in-part application of Ser. No. 09/081,380, filed May 19, 1998, now U.S. Pat. No. 6,086,935, and this application also is a continuation-in-part application of Ser. No. 08/898,736 filed Jul. 23, 1997. 
     
    
     
         [0002]    The present invention generally relates to a method for malting seeds, such as barley seeds and other cereals, such as wheat and sorghum seeds, legumes and pseudo-cereals. More particularly, the method includes reducing the microbial count of the seeds prior to malting and/or prior to adding a starter culture for the malting process.  
           [0003]    Heat and moisture are applied onto the particulate seeds to provide a heated moist environment which provides surface moisture on the seeds during the application of heat. Heat is applied for a time and temperature sufficient to reduce the microbial load of the seeds or other malting seeds without affecting or minimally affecting, the loss of the ability of the seeds to germinate and undergo a malting process.  
         BACKGROUND OF THE INVENTION  
         [0004]    Barley and other seeds, such as wheat and sorghum, are used for the production of beverages, such as beer. In most cases, a seed is subjected to a malting process to increase enzymatic activity. These enzymes, such as amylases, betaglucanase and xylanase enzymes, degrade the starch and non starchy components from the seeds to prepare them to be used in processes such as brewing In known malting processes, the moisture content of a seed, such as barley, is raised by immersion of the barley seeds in water. Thereafter, the barley with a high moisture content is allowed to germinate to provide a green malt. After germination, the green malt is dried under specific conditions. The final malt is cleaned to remove, for example, the sprouts and dust. Thereafter, it is used in processes, such as brewing, milling or other fermentation processes.  
           [0005]    Barley and seeds contain naturally occurring microorganisms. Generally, the quality of the malt and the beverage derived therefrom can be affected by the presence of microorganisms naturally present on and in the barley and other seeds. Some microorganisms positively affect malting, some do not. Generally, these microorganisms are further developed and grown during the malting process. During microorganism growth, metabolites can be produced, some of which are known to negatively influence the quality of the final malt. For example, some molds such as Fusarium can produce vomitoxin (=deoxynivalenol, often referred to as DON). DON is a harmful mycotoxin and levels of that mycotoxin should be kept as low as possible. It is believed mold or yeast strains developed or grown during the germination process also produce proteins which are claimed to cause gushing in beer produced from that malt. Moreover, other products formed by microorganisms during germination can be polysaccharides which are believed to cause early flocculation of yeast during fermentation in the brewing of the malted seeds. Hence, many microorganisms adversely affect the malting process and/or adversely affect the quality of the beverage which is made from the seeds, such as barley.  
           [0006]    Further, mycotoxins and/or other metabolites can be produced during the malting process. Indeed, malting process conditions from steeping to drying are such, that they encourage microbial growth and development and enhance the production of metabolites. And even though these metabolites originally present on the seed before the process generally are removed with the steep water after steeping, not all of the microorganisms and metabolites are removed Moreover, after steeping and during the germination process and the first phase of the drying process, microorganisms can again develop and produce these metabolites.  
           [0007]    A lower initial microbial count in seeds which undergo a malting process would not only reduce the level of undesirable microorganisms in the seeds, but also could prevent the production of metabolites of those microorganisms. A treatment lowering microbial count must be balanced so as not adversely affect the ability of the barley and other seeds to germinate, nor to destroy viable seed tissues critical to obtaining a good quality malted seeds. Hence, the embryo in the barley and other seeds should not be so adversely affected so that they will not be metabolically inactivated and will not germinate and produce, for example, enzymes as part of the malting process.  
         OBJECTS OF THE INVENTION  
         [0008]    An object of the invention is to provide an improved preparation process for making malted seeds and to improve the malted seeds product of such process.  
           [0009]    Another object of the invention is to provide a process and improved malted seed which vary less in quality with the raw material used.  
           [0010]    Additional objects of the invention will become apparent with reference to this specification.  
         SUMMARY OF THE INVENTION  
         [0011]    The invention provides a process or method of malting seeds selected from the group consisting of cereals, such as barley, wheat, rye, corn, oats, rice, millet and sorghum, pseudo-cereals, such as buckwheat, amaranth and legumes, such as soy. The process of the invention includes reducing the microbial level in the seed prior to steeping and malting and also can comprise the introduction of a starter culture used in the malting process. According to the invention, the reduction of microbial levels permits efficient use of starter cultures, such as molds, yeasts, bacteria, spores and activated spores. The invention also promotes malting without competition from microorganisms naturally present on the seeds before malting which not only negatively influence the malting process and the quality of the final malt, but also the quality of the products derived from the malted seeds In an important aspect, the invention provides a method for malting barley, sorghum and wheat, and in a very important aspect, barley. In another important aspect, the invention includes the addition of a starter culture, such as spores or activated spores, after the reduction of the microbial load. In the latter aspect, a starter culture, such as activated spores, provides increased enzymatic activity which is enhanced by reducing the microbial level in the seeds prior to the malting process The increase of enzymatic activity may be due to reduced competition between the starter culture and the natural occurring microorganisms which have been reduced during the treatment, as well as possible changes in the surface properties of the seeds that permit a better adhesion of a starter culture on the seeds. In yet another important aspect, the process contemplates a continuous treating of the seeds prior to the addition of the starter culture and continuation of the malting process.  
           [0012]    The method of the invention is unique in that it may be continuous and does not require increased pressures with sealed containers. The seeds are heated with moist heat and moisture on the surface of the seeds. Care should be taken not to over expose the seeds to the moist heat and/or over expose the seeds to moisture prior to heating because the seeds should not be cooked. Over exposing the seeds to moisture prior to heat will hydrate the seeds below the surface of the seeds and then the moist heat, such as steam, will tend to over heat the seeds, “cook” them and reduce their ability to germinate and sprout during the malting process. It is surface moisture on the surface of the seeds coupled with heating which is important in reducing the microbial content of the seeds without substantially adversely affecting the ability of the seeds to germinate and to produce enzymes which are sought through the malting process. Moist heat is applied by a fluid or fluidum which includes heated water or steam having a temperature in the range of about 60° C. to about 200° C.  
           [0013]    The method of the invention will reduce the microbial count of the seeds, such as barley and wheat, by at least about five (5) times per gram of product for molds and 1×10 2  per gram for yeasts, and in an important aspect by at least 1×10 3  per gram of product for molds and 1×10 4  per gram of product for yeasts. Further, the method of the invention is practiced without the necessity of pressurizing the heating vessel to pressures substantially above one atmosphere or atmospheric conditions.  
           [0014]    The method includes exposing the seeds to moist heat for a time and temperature that will reduce the microbial count, such as yeast and mold microbes, of the seeds by at least about a factor of five (5) per gram of seed, but will not reduce the germination energy of the seed after about 72 hours to less than about 80%. In general this means that seeds, such as barley and wheat, should be heated with surface moisture on the seeds for from about one to about thirty seconds at a temperature in the range of from about 50° C. to about 90° C. and preferably from about 60° C. to about 90° C. Temperature can be conveniently measured by placing a thermometer in the seed bulk. In one important aspect, the seed is a continuously moving stream of seeds. Where it is heated in a moist environment when it is a continuous moving stream, the seeds may be conveyed through a tube-like conduit via paddles where steam having a temperature of from about 100° C. to about 200° C. is injected through the paddles onto the seeds and increase the moisture content on the seeds. As the seeds go through the conduit, they have steam directly injected onto them which moisturizes and heats the surface of the seeds The important function of the steam is to provide an amount of surface moisture on the malting seeds which with the heat will kill undesirable microorganisms, but not adversely affect the ability of the seed to sprout and germinate. In this aspect, the steam is effective for heating the seeds to a temperature of at least about 50° C., and in an important aspect, heats the seeds from about 60 to about 90° C. as they leave the conduit, the seeds residing from about 1 to about 45 seconds, and preferably 3-30 seconds in the conduit. The conduit also may have a heat source (additional to the directly injected steam) which is a jacket which substantially surrounds the conduit throughout its length where the jacket is heated such as by steam, oil, electrically or any other suitable means for heating. The heated jacket indirectly heats the seed.  
           [0015]    In another important aspect of the invention where the seeds are treated when continuously moving as a stream, the seeds are conveyed on a pervious belt and has steam having a temperature of from about 100° C. to about 200° C. injected onto them as they proceed along the belt. In this aspect, the steam is applied to the surface of the seeds which are heated to at least about 50° C., and in an important aspect, the seeds are heated to about 65° C. to about 85° C. as they leave the belt, the seeds residing about 3 to about 30 seconds on the belt.  
           [0016]    In another aspect, the seeds may be immersed in water at about 65° C. to about 90° C. for about 1 to about 20 seconds for good results.  
           [0017]    The temperature of the heat source and time of exposure of the seeds to the heat source is a function of the sensitivity of the seeds to heating such that the time and temperature are effective for reducing the microbial count in the seeds by at least about a factor of five per gram of seeds, and the seed does not lose any substantial functionality for the malting process The combination of the temperature of the heat source, steam injection, surface moisture and time of exposure of the seeds to the heat source, however, should be effective to heat the seed to at least 50° C. and reduce the yeast and mold count by at least about a factor of five per gram of seed after the seeds are conveyed through the moist heat as described below.  
           [0018]    The process of the invention can also include cooling the seeds after heating the seeds with moist heat to cool the seeds down and to ambient temperature. The heating can permit prolonged storage of the seeds with reduced microbial count. 
       
    
    
     DESCRIPTION OF THE DRAWING  
       [0019]    [0019]FIG. 1 is a schematic diagram of an apparatus which can be used to practice the invention.  
         [0020]    [0020]FIG. 2 is a side view of the conduit in the apparatus shown in FIG. 1. 
     
    
     DETAILED DESCRIPTION  
     Definitions  
       [0021]    As used herein, the term “spore” refers to a dormant and highly resistant reproductive cell formed by bacteria and fungi in response to environment conditions that do not favor the growth of the organism. When exposed to favorable environmental conditions, spores are capable of developing into a viable adult organism without fusion with another cell.  
         [0022]    As used herein, the term “activated spore” means a spore having one of the following properties.  
         [0023]    I. The spore is swollen such that its size is increased by a factor of between about 1.2 and about 10 over its dormant size; and/or  
         [0024]    II. One or more germ tubes per spore is formed. Activated spores are prepared by one or a combination of the following treatments:  
         [0025]    i. Cycles of wetting and/or drying;  
         [0026]    ii. Addition of appropriate nutritional supplies (such as a nitrogen source, preferably amino acids and/or a carbon source, preferably mono-or disaccharides) or spore elements;  
         [0027]    iii. Exposure to temperature changes, preferably within a temperature range of about 0° to about 80° C.  
         [0028]    iv. Exposure to changes in pH, preferably within a pH range of about 2.0 to about 8.0,  
         [0029]    more preferably about 3.0 to about 6.0.  
         [0030]    The term “germination” as used herein means the beginning or resumption of growth by a seed. In accordance with the process of the present invention, germination begins to occur during and/or after the seeds have been steeped. Germination of seed is generally understood to mean hydration of the seeds, swelling of the seeds and inducing growth of the embryo. Environmental factors affecting germination include moisture, temperature and oxygen level. Root and shoot development are observed.  
         [0031]    As used herein, the term “steeping” refers to wetting of the seeds. Wetting may include one or more stages over a time and temperature effective for providing a moisture content of between about 20% and about 60% by weight.  
         [0032]    In one embodiment of the invention, the seeds for malting are fed into a elongated heating device  4  which is shown in FIG. 1 The seeds are fed into the heating device from hopper  6  through feed aperture  8  into channel  10 . The seeds are conveyed down channel  10  in the y direction. Channel  10  is surrounded by a steam jacket  12  through which steam may be circulated. A hollow rod  14  extends longitudinally down the center of the channel. A plurality of paddles  16  are mounted on the rod  14  down its longitudinal length. The rod  14  is rotated and the paddles are angled such that as the rod rotates the paddles, the paddles mix the seeds and push the seed down channel  10 . The paddles have openings  18  from channels which extend through the paddles to the hollow center of rod  14 . These openings transmit steam going through the rod and paddles so that the steam may be injected onto the seeds being transmitted down channel  10 . As the rod rotates, the paddles push the seed down the conduit to exit aperture  20  through which the seeds with the microbial reduced load drops. The openings in the paddles may be opened or closed to control steam injection onto the seeds being transmitted down the channel. In one aspect of the invention, some of the openings in the paddles towards the feed input end of the channel are open to transmit steam from the hollow rotating rod  14  onto the seeds. Steam can be supplied through the rod and through the paddles onto the to-be-treated particulate seeds. The amount of steam injected is effective for providing a moist environment, increasing the moisture content on the surface of the seeds and to heat the product as described above. Additional indirect heating of the particulate seeds may be done by using indirect heat from the jacket of the device. Just enough steam is injected to provide moisture on the surface of the to be treated particulate seeds. With the moisture and heat from the steam and the indirect heat from the jacket heat source of the device, the conditions are sufficient to kill microorganisms at the surface of the seeds without substantially reducing the ability of the seeds to germinate. The higher moisture content, the heat and the time of exposure of the seeds to the heat must be limited to prevent the functional properties of the cereal to be negatively influenced. A device which can be used to treat the particulate seed as described herein is available as a Solidaire Model SJCS 8-4 from the Hosokawa Bepex Corporation, 333 N.E. Taft Street, Minneapolis, Minn. 55413, but the invention is not limited to such a device or a device with paddles for the injection of steam.  
         [0033]    The rotating paddles as vehicles for injecting steam, assure that the steam is uniformly distributed over the surfaces of the seeds, but not into the seed particle. It is important that there is sufficient mixing of the seeds during the treatment that the surfaces of the seeds are moist and achieve a temperature of at least about 50° C.  
         [0034]    The starter culture, such as spores and/or activated spores, can be introduced before or during the various germination or steeping stages. For example, activated spores can be introduced during the various germination or steeping stages. In the case of activated spores, the concentration of the spores vary depending on the conditions of the malting process and the type of active spore being utilized. Generally about 1×10 2  to about 1×10 7 , preferably about 1×10 3  to about 1×10 5  activated spores per gram air dry seed is utilized.  
         [0035]    The seed after the steam and heat treatment then is wetted and mixed with a starter culture which may be combined with the seeds either before or after wetting. When the invention utilizes a starter culture, such as spores or activated spores from microoganisms, such as bacteria or molds, a surprising increase in enzymatic activity of the malted seeds results. In this aspect, the process generally comprises combining water, the seeds and activated spores and holding the combination until seeds of enhanced enzymatic activity is formed. Generally, the combination is made by inoculating the moistened seeds with the activated spores, but as previously noted, the spores or activated spores and seeds may be combined before or after the moistening of the seeds, but after reducing the microbial count of the seeds. In the process of the invention, the combination of wetted seeds and starter culture has a concentration of starter culture, holding time and holding temperature which are effective for providing the seeds with an increase in enzymatic activity of at least one enzyme, such as β-glucanase, xylanase, amylases, debranching enzymes, proteases and/or other naturally occurring enzyme activities, which are greater than the enzymatic activity which is obtained by holding the wetted seeds without the reduced microbial count and without a starter culture, such as activated spores.  
         [0036]    In this aspect, the seed, spores or activated spores are combined before or after the time of wetting the seeds and the combination is held at a temperature of at least about 5 20  C. and not more than about 30° C., preferably between about 10° C. to about 20° C. and the activated spores are at a concentration in the combination to obtain an increase in enzymatic activity of the malted seeds. The wetted or moistened seeds and activated spore combination is held for a time and temperature until the seeds have a moisture content of at least about 20 weight percent. The moistened seeds and activated spores are held together until the seeds have a moisture content of between about 20 to about 60 weight percent, preferably from about 38 to about 47 weight percent, and are allowed to germinate for about 2 to about 7 days, preferably about 3 to about 6 days, at a temperature of from about 10° to about 30° C., preferably from 14° to about 21° C. In important aspect, the germinated seeds are dried at a temperature of from about 40° to about 150° C., preferably between about 45° and 85° C. until the dried malted seeds have a moisture content of from about 2 to about 15 weight percent moisture, preferably from about 3 to about 7 weight percent moisture.  
         [0037]    Microorganisms, spores, and activated spores which may be used in the invention may be from the microbes selected from the group comprising of Enterococcus spp., Micrococcus spp., Pediococcus spp., Leuconostoc spp., Lactobacillus spp., Brevibacterium spp., Corynebacterium spp., Propionibacterium spp., Bacillus spp., Acetobacter spp., Pseudomonas spp., Pichia spp., Sacchaaromyces spp., Zygosaccharomyces spp., Hanseniaspora spp., Rhodotorula spp., Torulopsis spp., Trichosporon spp, Kloeckera spp., Candida spp., Geotrichum spp., Neurospora spp., Monascus spp., Mucor spp., Rhizopus spp., Rhizopus oryzae strain ATCC 9363, Trichoderma spp., Aspergillus spp., Penicillium spp., Amylomyces spp., and mixtures thereof. The scope of the present invention is, however, not limited to the above-mentioned list of microorganisms.  
       EXAMPLE 1—FLUIDIZED BED SYSTEM  
       [0038]    Barley was used in the test. Samples of barley were placed in a special device or chamber. Heated moist air was injected into the barley samples. This injection resulted in a fluidization of the barley and a surface treatment to reduce the microbial count. The time is the time in seconds the barley was treated in the chamber. The temperature is the temperature of the barley sample direct after treatment. The barley treated was a US 6-row variety standard used for malting purposes. Samples were analyzed on microbial count such as total aerobic plate count, molds and yeasts. The germination energy was measured according to the B.F. method 3.6.2 Analytic EBC with the addition of 4 ml water. Germination energy of 100 means that all the barley analyzed was germinated after 72 hours.  
                                                                                                 Germ                       Time       Energy   T.P.C./   Yeasts/   Molds/       Test   (sec)   Temp (C.)   72 hrs.   Gram   Gram   Gram                                1   3   67   93   5.20E+05   100   2,900         1b   3   66   92   4.50E+05   350   2,300         2   3   77   90   3.50E+05   20   1,700         3   3   77   90   1.00E+05   200   1,400         4b   3   85   95   2.00E+06   &lt;10   630       4   7   85   90   7.40E+05   &lt;10   210       5   7   67   90   1.60E+06   30   310       7   7   77   98   1.20E+06   &lt;10   320       8   7   87   70   5.00E+06   &lt;10   170       9   14   67   85   1.80E−06   &lt;10   550       9b   14   67   87   1.40E+06   &lt;10   290       10   14   78   83   1.00E+06   &lt;10   160       11   14   78   71   9.30E+04   &lt;10    60       12   14   85   40   5.60E+03   &lt;10    40       13   28   67   65   1.50E+04   &lt;10   580       14   28   77   25   6.00E+02   &lt;10    10       15   28   78   15   8.00E+02   &lt;10    20       16   28   85   35   1.10E+03   &lt;10   650       16b   28   84   15   9.70E+03   10    20       control           95   7.60E+07   34,000   26,000                    
 
       EXAMPLE II—ROTOR WITH PADDLES  
       [0039]    Barley: samples treated with a rotor which includes paddles which inject steam onto the barley being conveyed down a tunnel-like conduit by the rotating paddles.  
         [0040]    Control: not steam heat treated.  
         [0041]    Sample: steam heat treated.  
         [0042]    The barley treated was a 6-row malting barley variety as described in Example I.  
         [0043]    Microbial Reduction Treatment: product temperature 60° C., time treatment in the conduit: 10 seconds; direct steam temperature 111° C. Jacket temperature (indirect) 149-150° C.  
         [0044]    Microbial content sample:  
                                                                             T.P.C./g   Yeasts/g   Molds/g                                        Control   670000   35000   27000           Sample    50000    100    2400                      
 
         [0045]    Malting process: the microbial reduced barley (400 g) was steeped in an erlemeyer flask (2 liter). Barley: water ratio 1:1. The erlemeyer was agitated on an orbital shaker (100 rpm) during the wet stages. During the wet stages the barley was aerated with filter sterilized air. The steeping program was carried out by:  
         [0046]    wet stage, 6 hrs.  
         [0047]    dry stage, 17 hrs.  
         [0048]    wet stage 5 hrs.  
         [0049]    dry stage 16.50 hrs.  
         [0050]    wet stage 2.50 hrs.  
         [0051]    After steeping, the germination was carried out in a Joe White micromalting unit using three temperature steps: 14 hours at 20° C., 2 hours at 18° C., 80 hours at 16° C. Kilning was carried out in the same malting unit using 7 temperature steps: 3 hours at 62° C., 2 hours at 65° C., 2 hours at 68° C., 2 hours at 73° C., 1 hour at 78° C., 2 hours at 80° C, 6 hours at 83° C,  
         [0052]    DON analysis were done using a GC method.  
         [0053]    Malting with a starter culture (S.C.): Activated spores from Rhizopus oryzae ATCC 9363 were used as starter culture. Activated spores (10.000/per gram dry barley) were added during the first wet steeping cycle.  
                                                                             DON Analysis on Malted Barley                DON ppm                    Average   st. dev.                            Barley   3.34   0.69           Malt           Control   0.53   0.69           Sample   0.27   0.01           Control with S.C.   0.43   0.11           Sample with S.C.   0.36   0.05                      
 
         [0054]    2 trials, 2 analyses each  
         [0055]    Results show that the steam treated barley gives a lower DON value than the untreated barley and that the standard deviation of the treated barley is much lower.  
         [0056]    Results: beta glucanase activity (abs/g dry weight) in the dried malt.  
                                                                             1   2   3                                        Blanc       17.78   11.89           Test       13.06   8.57           Blanc with S.C.   64.57   30.96   85.78           Test with S.C.   156.3   138.15   189.12                      
 
         [0057]    Results show that the steam treated barley gives with the starter culture an increased level of enzyme activity.  
       EXAMPLE III—IMMERSION BARLEY  
       [0058]    Barley samples were immersed for different times in water at different temperatures. After immersion, the samples were dried and further analyzed. The barley was 6 Row U.S. barley as described in Example I.  
                                                         BARLEY                    Product   Germ. Energy               Test   Time (s)   Temp (C.)   72 hr   T.P.C./g   Molds/g               1   2 sec   95 C.   84    7300    110       2   2 sec   95 C.   89    96000   96000        3   5 sec   95 C.   52     800    800       4   5 sec   95 C.   50     70    70       5   2 sec   75 C.   89   3200000     300       6   2 sec   75 C.   89   300000   2100       7   5 sec   75 C.   90   190000   1100       8   7 sec   75 C.   86   150000    210       9   7 sec   75 C.   93   470000    150       10    14 sec    75 C.   90    50000   1000       11    Ctrl   —   94   190000   99000                   
 
       EXAMPLE IV—IMMERSION WHEAT  
       [0059]    Wheat samples were immersed for different times in water at different temperatures. After immersion, the samples were dried and further analyzed. The wheat was Stephen a White wheat variety, used for malting purposes.  
                                                                           Germ.   T.P.C./                           Temp.   Energy   Grams   Yeasts/   Molds/       Sample   Test   Time(s)   C.   % *   **   Grams   Grams                   Wheat   1   3   66   100    1.60E+05   1.00E+01   3.60E+02       Wheat   1b   3   66   100    1.20E+05   &lt;10   1.60E+02       Wheat   2   3   77   100    1.50E+05   &lt;10   1.50E+02       Wheat   3   3   77   97   1.80E+05   &lt;10   7.00E+01       Wheat   4   3   88   78   4.90E+04   &lt;10   4.00E+01       Wheat   4b   3   88   75   9.00E+05   &lt;10   1.70E+02       Wheat   5   7   66   100    1.80E+05   &lt;10   2.10E+03       Wheat   6   7   77   93   6.40E+04   &lt;10   1.20E+02       Wheat   7   7   77   92   1.50E+05   &lt;10   1.00E+03       Wheat   8   7   88   15   8.30E+03   &lt;10   &lt;10       Wheat   9   Ctrl   —   100    1.30E+05   4.40E+03   1.00E+03                                  
 
       EXAMPLE V—BARLEY  
       [0060]    6-Row U.S. malting barley was treated in a conduit with direct and indirect heat. After the treatment, samples were cooled and dried to remove surface moisture. Samples were further analyzed.  
                                                                                             Direct                       Indirect   Steam   Temp. of                   Heat   Temp.   Product       Germination           Jacket   from   after   Time   Energy           Temp.*   Rotor*   Treatment*   (sec.)   72 hrs.                                    Test a   241   251   155   7.5   80       Test b   281   251   161   7.5   88       Test c   251   299   165   10.5   81       Control                   91                          
 
         [0061]    [0061]                                                                             T.P.C./gram   Yeasts/gram   Molds/gram                                        Test a   3.00E+06   1.00E+01   7.00E+02           Test b   1.60E+06   &lt;10   5.00E+01           Test c   1.40E+07   &lt;10   &lt;10           Control   5.70E+06   2.10E+04   1.30E+04