Abstract:
This disclosure relates to enhancing growth and/or activity of lactobacilli using a prebiotic formulation which includes iso-malto oligosaccharides and α-galactosidase; and to enhancing growth and/or activity of bifidobacteria using a prebiotic formulation which includes iso-malto oligosaccharides and β-glucanase. Other combinations of fibers and enzymes are described below which also stimulate growth and activity of lactobacilli or bifidobacteria. These combinations of enzymes and prebiotics can be taken separately or added to foods, including desserts.

Description:
FIELD OF THE APPLICATION 
     The field is enzymes and prebiotics for enhancing probiotic efficacy. 
     BACKGROUND 
     Lactic acid producing bacteria that are capable of improving or maintaining intestinal health and function, including reducing constipation (primarily from the  Lactobacillus  and  Bifidobacterium  genera) are termed probiotic bacteria. Dietary supplements with probiotic bacteria as the active ingredient currently enjoy sales of over $700 million annually, and the market growth is approaching 30% annually. The other piece of the probiotic market is probiotic foods, especially yogurt and desserts. This segment of the market is over $1 Billion annually. 
     The reported health benefits of probiotics include supporting the immune system (inhibiting allergic response and neoplastic growth), treating inflammatory bowel disease, offsetting lactose intolerance, and reducing cholesterol. They are also useful for repopulating the gut after antibiotic therapy. Probiotic growth in the intestinal tract, following ingestion, depends to a large extent on the nutrients present in the patient&#39;s diet. Typical human diets are not well suited for probiotics and, given the abundance of and competition from many less fastidious digestive tract bacteria (including pathogenic strains such as clostridium, rotaviruses, pathogenic  E. coli  and  Helicobacter pylori ) it can be difficult for probiotics to effectively multiply in vivo. To help correct this problem, manufacturers of probiotic dietary supplements often include prebiotics (nutrient substances that encourage the growth of probiotics in vivo) in their formulations. 
     Many types of prebiotics are not digested or absorbed in the small intestine but pass into the colon where they stimulate the growth of probiotic bacteria. Fructo-oligosaccharides (FOS) are one type of prebiotic; inulin compounds (which are also oligosaccharides) are another. For these compounds to be effective they must be ingested in relatively large quantities, such as 4-10 grams/day for FOS and 10-14 grams/day for inulin. Probiotics, by comparison, can be effectively administered in milligram quantities, containing 10 7 -10 10  colony forming units (cfu). Thus, it is impractical to mix FOS or inulin with probiotics and deliver them in capsules or tablets. Further, such carbohydrate type prebiotics often break down to glucose, in vivo, which enhances growth of non-probiotic bacteria, including pathogenic clostridium. Moreover, FOS can cause flatulence and abdominal pain and some people experience severe allergic reactions to inulin. Therefore, there is a need for a non-carbohydrate prebiotic that can be used at low dosage while effectively stimulating probiotic bacteria. 
     Although enzymes have been used to generate prebiotics under laboratory conditions followed by subsequent feeding of the preformed prebiotics to achieve probiotic stimulation (see U.S. Pat. Nos. 6,791,015 and 6,730,502), no one has suggested using enzymes to generate these effects in vivo. U.S. Pat. No. 5,817,350 discloses the use of the prebiotic enzymes cellulase, amylase and hemicellulase, for use as dietary supplements, but not use of these enzymes to stimulate administered probiotics. Enzymes which can generate compounds which significantly increase probiotic growth or activity without generating significant amounts of glucose or otherwise stimulating growth of undesirable digestive tract bacteria, would be a significant improvement over existing formulations. 
     Iso-malto oligosaccharides can be enzymatically digested to simpler sugars by inulinase, which is included in some commercially-available probiotic formulations because it digests linear fructans (inulin). Inulin is known to stimulate bifidobacteria growth. Inulin in diet does not lead to a rise in serum glucose or stimulate insulin secretion, but inulinase digestion generates significant fructose. It is not clear whether fructose would preferentially increase growth of probiotics or of competitive digestive tract bacteria, including pathogenic bacteria. 
     The product Beano™ includes the enzyme alpha-galactosidase, which can break down polysaccharides and oligosaccharides, including iso-malto oligosaccharides, which are in foods such as legumes (beans and peanuts) and cruciferous vegetables (cauliflower, broccoli, cabbage, brussels sprouts, among others). The enzyme breaks those complex sugars into simpler sugars, making these foods somewhat more digestible, and thereby reducing intestinal gas. Beano does not include any probiotics in its formulation. 
     The hydrolysis of lactose to glucose and galactose is catalyzed by the enzymes lactase and β-galactosidase. Because β-galactosidase would generate glucose from lactose in the diet, it is not preferred for inclusion in probiotics.  Lactobacillus bulgaricus  produces beta-galactosidase, and this strain is a probiotic purported to treat lactose intolerance. 
     SUMMARY 
     This disclosure relates to enhancing growth and/or activity of lactobacilli using a prebiotic formulation which includes iso-malto oligosaccharides and α-galactosidase; and to enhancing growth and/or activity of bifidobacteria using a prebiotic formulation which includes iso-malto oligosaccharides and β-glucanase. Other combinations of fibers and enzymes are described below which also stimulate growth and activity of lactobacilli or bifidobacteria. 
     The enzymes α-galactosidase and β-glucanase react with the fiber prebiotics to generate shorter chain oligosaccharides, some of which are preferential growth enhancers for the probiotics. The enzymes are believed to not generate significant amounts of glucose in the reaction, as it can stimulate growth of undesirable bacterial species. 
     The fiber prebiotic, the enzyme(s) and the probiotic(s) can be administered in a combined formula, or, the fiber prebiotic with the appropriate enzyme (e.g., α-galactosidase or β-glucanase) can be in media where they can react (e.g., added to foods) and the probiotic can be administered separately. Or, each of these ingredients can be administered separately, whereby the prebiotic and the enzyme can react in vivo, and the probiotic can metabolize the reaction product(s) to enhance its growth and activity. 
     More specifically, the invention relates to enhancing in vivo growth and/or activity of both lactobacilli and bifidobacteria using iso-malto oligosaccharides as the prebiotic, and both α-galactosidase and β-glucanase as the enzymes. Again, these ingredients can be combined or administered separately. 
     Pectinase may also be included in any of the formulations described herein. Pectinase are a class of enzymes including pectolyase, pectozyme and polygalacturonase. They break down pectin, a polysaccharide found in the cell walls of plants. 
     The above formulas could also include other prebiotics (including inulin, wheat dextrin, and partially hydrolyzed guar gum (“PHGG”)) and other fiber-digesting enzymes, including Fiberase™ (a combination of cellulase, hemicellulase, pectinase and xylanase). Cellulase includes cellulase-TL and cellulase-AN. The formulas could also include protease enzymes including papain, bromelain, fungal protease, fungal acid-protease, bacterial protease, fungal peptidase, nattokinase, serapeptase, trypsin, chymotrypsin pancreatin and pepsin. Carbohydrase enzymes (including alpha-amylase, amylase, glucoamylase, lactase, and invertase) are generally not preferred in the formula, as they generate glucose. 
     The above formulas could also include sunflower lecithin and/or oleic acid (as described in U.S. Pat. No. 8,105,577, incorporated by reference) and/or the food grade polysorbate surfactants (as described in U.S. Pat. No. 8,066,986, incorporated by reference): Polysorbate-60, polysorbate-80 or any polysorbate with an HLB&gt;12, where HLB is the hydrophile-lipophile balance, designated from 1 to 20. 
     The above formulas could also contain other carriers, binders or adsorbents, including but not limited to food grade starches and silicates. The above formulas can be packaged for administration in capsules, tablets or packets, or combinations thereof. Alternatively, they can be added to foods, separately or in combination. 
     Additional combinations of substrates, enzymes and probiotics which enhanced growth and/or activity of the probiotics are described below and are also within the scope of the inventions herein. 
    
    
     
       BRIEF DESCRIPTION OF THE DRAWINGS 
         FIG. 1A  shows the growth of BL-04 ( Bifidobacterium lactis ) in MRS broth (“Control (scratch)”) made by the experimenter to have the same constituents as commercial MRS broth (by Difco™), and its growth in modified MRS broth where the glucose has been removed and a quantity of fiber (in this case, VitaFiber™, by BioNeutra) has been added which is approximately the same as the amount of glucose removed (hereinafter, such modified MRS broth is referred to as “No-G-Broth”). 
         FIG. 1B  shows the activity of BL-04 ( Bifidobacterium lactis ) in MRS broth, and in No-G-Broth with added VitaFiber™ 
         FIG. 2A  shows the growth of NCFM ( Lactobacillus acidophilus ) in MRS broth, and in No-G-Broth with added VitaFiber™ as in  FIGS. 1A ,  1 B. 
         FIG. 2B  shows the activity of NCFM ( Lactobacillus acidophilus ) in MRS broth, and in No-G-Broth with added VitaFiber™ as in  FIGS. 1A ,  1 B. 
         FIG. 3A  shows the growth of BL-04 ( Bifidobacterium lactis ) in No-G-Broth with added VitaFiber™ and where VitaFiber™ was pre-digested with cellulase before the remaining No-G-Broth broth ingredients were added. 
         FIG. 3B  shows the activity of BL-04 ( Bifidobacterium lactis ) in No-G-Broth with added VitaFiber™, and where VitaFiber™ was pre-digested with cellulase before the remaining No-G-Broth broth ingredients were added. 
         FIG. 4A  shows the growth of NCFM ( Lactobacillus acidophilus ) in No-G-Broth with added VitaFiber™, and where VitaFiber™ was pre-digested with pectinase before the remaining No-G-Broth broth ingredients were added. 
         FIG. 4B  shows the activity of NCFM ( Lactobacillus acidophilus ) in No-G-Broth with added VitaFiber™, and where VitaFiber™ was pre-digested with pectinase before the remaining No-G-Broth broth ingredients were added. 
         FIG. 5A  shows the growth of BL-04 ( Bifidobacterium lactis ) in No-G-Broth with added VitaFiber™, and where VitaFiber™ was pre-digested with either β-glucanase or α-galactosidase before the remaining No-G-Broth broth ingredients were added. The Vitafiber™ was sterilized by filtering it through a sterile 0.22 μm filter, while the MRS broth ingredients in the VitaFiber™ mixture were separately sterilized by autoclaving. 
         FIG. 5B  shows the activity of BL-04 ( Bifidobacterium lactis ) in the same conditions and media as in  FIG. 5A . 
         FIG. 6A  shows the growth of NCFM ( Lactobacillus acidophilus ) in No-G-Broth with added VitaFiber™, and where VitaFiber™ was pre-digested with either β-glucanase or α-galactosidase before the remaining No-G-Broth broth ingredients were added. 
         FIG. 6B  shows the activity of NCFM ( Lactobacillus acidophilus ) in the same conditions and media as in  FIG. 6A . 
         FIG. 7A  shows the growth of BL-04 ( Bifidobacterium lactis ) in No-G-Broth where a quantity of partially hydrolyzed guar gum (“PHGG”) has been added which is approximately the same as the amount of glucose removed from the starting MRS broth; and where PHGG was pre-digested with either β-glucanase or α-galactosidase before the remaining No-G-Broth broth ingredients were added. 
         FIG. 7B  shows the activity of BL-04 ( Bifidobacterium lactis ) in the same conditions and media as in  FIG. 7A . 
         FIG. 8A  shows the growth of NCFM ( Lactobacillus acidophilus ) in No-G-Broth where a quantity of partially hydrolyzed guar gum (“PHGG”) has been added which is approximately the same as the amount of glucose removed from the starting MRS broth; and where PHGG was pre-digested with either β-glucanase or α-galactosidase before the remaining No-G-Broth broth ingredients were added. 
         FIG. 8B  shows the activity of NCFM ( Lactobacillus acidophilus ) in the same conditions and media as in  FIG. 8A . 
         FIG. 9A  shows the growth of LP-115 ( Lactobacillus plantarum ) in No-G-Broth with added VitaFiber™, and where VitaFiber™ was pre-digested with either with either pectinase or a 50:50 mixture of α-galatosidase plus pectinase before the remaining No-G-Broth ingredients were added. Note that “cold filter” means the VitaFiber™ was sterilized by filtering it through a sterile 0.22 μm filter, while the MRS broth ingredients in the VitaFiber™ mixture were separately sterilized by autoclaving. 
         FIG. 9B  shows the activity of LP-115 ( Lactobacillus plantarum ) in the same conditions and media as in  FIG. 9A . 
         FIG. 10A  shows the growth of NCFM ( Lactobacillus acidophilus ) where VitaFiber™ was pre-digested with α-galactosidase before the remaining No-G-Broth ingredients (except polysorbate 80) were added along with added LactoStim™ (sunflower lecithin and oleic acid). VitaFiber™ was also pre-digested with α-galactosidase plus pectinase, before the remaining No-G-Broth ingredients were added. 
         FIG. 10B  shows the activity of NCFM ( Lactobacillus acidophilus ) in the same conditions and media as in  FIG. 10A . 
         FIG. 11A  shows the growth of BL-04 ( Bifidobacterium lactis ) where VitaFiber™ was pre-digested with β-glucanase before the remaining No-G-Broth ingredients (except polysorbate 80) were added along with added LactoStim™, and where VitaFiber™ was pre-digested with β-glucanase and cellulase before the remaining No-G-Broth ingredients were added. 
         FIG. 11B  shows the growth of BL-04 ( Bifidobacterium lactis ) in the same conditions and media as in  FIG. 11A . 
         FIG. 12A  shows the growth of BL-04 ( Bifidobacterium lactis ) in No-G-Broth where a quantity of inulin has been added which is approximately the same as the amount of glucose removed from the starting MRS broth, and where inulin was pre-digested with β-glucanase before the remaining No-G-Broth ingredients were added. 
         FIG. 12B  shows the activity of BL-04 ( Bifidobacterium lactis ) in the same conditions and media as in  FIG. 12A . 
         FIG. 13A  shows the growth of NCFM ( Lactobacillus acidophilus ) in No-G-Broth with inulin substituted for glucose, and where inulin was pre-digested with α-galactosidase before the remaining No-G-Broth ingredients (except polysorbate 80) were added along with added LactoStim™. 
         FIG. 13B  shows the activity of NCFM ( Lactobacillus acidophilus ) in the same conditions and media as in  FIG. 13A . 
         FIG. 14A  shows the growth of BL-04 ( Bifidobacterium lactis ) in No-G-Broth with VitaFiber™ substituted for glucose, and where VitaFiber™ was pre-digested with one of two different concentrations of β-glucanase before the remaining No-G-Broth ingredients were added. 
         FIG. 14B  shows the activity of BL-04 ( Bifidobacterium lactis ) in the same conditions and media as in  FIG. 14A . 
         FIG. 15A  shows the growth of NCFM ( Lactobacillus acidophilus ) in No-G-Broth with VitaFiber™ substituted for glucose, and where VitaFiber™ was pre-digested with one of: (i) α-galactosidase; (ii) a lower concentration of α-galactosidase and β-glucanase; and (iii) a higher concentration of α-galactosidase and β-glucanase. 
         FIG. 15B  shows the activity of NCFM ( Lactobacillus acidophilus ) in the conditions and same media as in  FIG. 15A . 
         FIG. 16A  shows the growth of BL-04 and NCFM (i) in MRS broth, (ii) in No-G-Broth with VitaFiber™ substituted for glucose, (ii) where VitaFiber™ was pre-digested with a 1:3 blend of 3-glucanase and α-galactosidase before (a) adding the other ingredients in No-G-Broth, (b) adding LactoStim™ and the other ingredients in No-G-Broth but not polysorbate 80. 
         FIG. 16B  shows the activity of BL-04 and NCFM in the same conditions and media as in  FIG. 16A . 
         FIG. 17A  shows the growth of Latobacillus  salivarius  (LS-33) where VitaFiber™ was pre-digested with one of: (i) α-galatosidase; (ii) β-glucanase; and (iii) a 1:3 blend of β-glucanase and α-galatosidase; before adding LactoStim™ and the other ingredients in No-G-Broth but not polysorbate 80. 
         FIG. 17B  shows the activity of Latobacillus  salivarius  (LS-33) in the same conditions and media as in  FIG. 17A . 
         FIG. 18A  shows the growth of  Lactobacillus paracasei  (LPC-37) where VitaFiber™ was pre-digested with one of: (i) α-galatosidase; (ii) β-glucanase; and (iii) a 1:3 blend of β-glucanase and α-galatosidase, before adding LactoStim™ and the other ingredients in No-G-Broth but not polysorbate 80. 
         FIG. 18B  shows the activity of  Lactobacillus paracasei  (LPC-37) in the same conditions and media as in  FIG. 18A . 
         FIG. 19A  shows the growth of  Lactobacillus plantarum  (LP-115) where VitaFiber™ was pre-digested with one of: (i) α-galatosidase; (ii) β-glucanase; and (iii) a 1:3 blend of β-glucanase and α-galatosidase before adding LactoStim™ and the other ingredients in No-G-Broth but not polysorbate 80. 
         FIG. 19B  shows the activity of  Lactobacillus plantarum  (LP-115) in the same conditions and media as in  FIG. 19A . 
         FIG. 20A  shows the growth of  Lactobacillus rhamnosus  (Lr-32) where VitaFiber™ was pre-digested with one of: (i) α-galatosidase; (ii) β-glucanase; and (iii) a 1:3 blend of β-glucanase and α-galatosidase before adding LactoStim™ and the other ingredients in No-G-Broth but not polysorbate 80. 
         FIG. 20B  shows the activity of  Lactobacillus rhamnosus  (Lr-32) in the same conditions and media as in  FIG. 20A . 
         FIG. 21A  shows the growth of  Bifidobacterium lactis  (Bi-07) where VitaFiber™ was pre-digested with one of: (i) α-galatosidase; (ii) β-glucanase; and (iii) a 1:3 blend of β-glucanase and α-galatosidase, before adding LactoStim™ and the other ingredients in No-G-Broth but not polysorbate 80. 
         FIG. 21B  shows the activity of  Bifidobacterium lactis  (Bi-07) in the same conditions and media as in  FIG. 21A . 
         FIG. 22A  shows growth of  Lactobacillus rhamnosus  (LR-32) with Wheat Dextrin in No-G-Broth, and where Wheat Dextrin was pre-digested with α-galatosidase before adding the other ingredients in No-G-Broth. 
         FIG. 22B  shows the activity of  Lactobacillus rhamnosus  (LR-32) in the same conditions and media as in  FIG. 22A . 
         FIG. 23A  shows the growth of  Lactobacillus salivarius  (LS-33) with Wheat Dextrin in No-G-Broth, and where Wheat Dextrin was pre-digested with α-galatosidase before adding the other ingredients in No-G-Broth. 
         FIG. 23B  shows the activity of  Lactobacillus salivarius  (LS-33) in the same conditions and media as in  FIG. 23A . 
         FIG. 24A  shows the growth of  Lactobacillus acidophilus  (NCFM) with Wheat Dextrin in No-G-Broth, and where Wheat Dextrin was pre-digested with α-galatosidase before adding the other ingredients in No-G-Broth. 
         FIG. 24B  shows the activity of  Lactobacillus acidophilus  (NCFM) in the same conditions and media as in  FIG. 24A . 
         FIG. 25A  shows the growth of  Bifidobacterium lactis  (BL-04) with Wheat Dextrin in No-G-Broth, and where Wheat Dextrin was pre-digested with β-glucanase before adding the other ingredients in No-G-Broth. 
         FIG. 25B  shows the activity of  Bifidobacterium lactis  (BL-04) in the same conditions and media as in  FIG. 25A . 
         FIG. 26A  shows the growth of  Bifidobacterium lactis  (Bi-07) with Wheat Dextrin in No-G-Broth, and where Wheat Dextrin was pre-digested with β-glucanase before adding the other ingredients in No-G-Broth. 
         FIG. 26B  shows the activity of  Bifidobacterium lactis  (Bi-07) in the same conditions and media as in  FIG. 26A . 
         FIG. 27A  shows the growth of  Bifidobacterium breve  (BB-03) with Wheat Dextrin in No-G-Broth, and where Wheat Dextrin was pre-digested with β-glucanase before adding the other ingredients in No-G-Broth. 
         FIG. 27B  shows the activity of  Bifidobacterium breve  (BB-03) in the same conditions and media as in  FIG. 27A . 
         FIG. 28A  shows the growth of  Lactobacillus plantarum  (LP-115) with Wheat Dextrin in No-G-Broth, and where Wheat Dextrin was pre-digested with either pectinase or α-galactosidase before adding the other ingredients in No-G-Broth. 
         FIG. 28B  shows the activity of  Lactobacillus plantarum  (LP-115) in the same conditions and media as in  FIG. 28A . 
     
    
    
     DETAILED DESCRIPTION 
     Certain enzymes, acting upon certain fiber sources, render the fiber sources a preferential food source (prebiotic) for probiotic bacteria. As shown in the figures, different species of probiotics, all of which are lactic acid producing bacteria, respond differently to various enzymes and fiber sources. All of the enzymes described are “fiber-digesting” enzymes, which render complex oligosaccharides into simpler oligosaccharides, but without significant production of glucose. Particular enzyme/fiber combinations respectively improve both activity and growth of  Lactobacillus  and of Bifidobacteria. 
     These combinations of enzymes and prebiotics can be used to improve the commercial value and performance of probiotic products, which all include  Lactobacillus  and/or Bifidobacteria. These combinations of enzymes and prebiotics can be formulated with  Lactobacillus  and/or Bifidobacteria, e.g., in a capsule or tablet form. Another use for them would be as food additives to foods that do not requiring heating/boiling before consumption, e.g., yogurt, ice cream, desserts, bread or other bakery goods, snacks, breakfast cereal or candy. 
     These combinations of enzymes and prebiotics could be added to such foods with or without probiotics, and with or without other growth stimulants for probiotics (e.g., polysorbate 80, sunflower lecithin or oleic acid). If such combinations of enzymes and prebiotics were added, for example, to yogurt, they could act to produce the less complex oligosaccharides after consumption. If the probiotics (with or without other growth stimulants) are ingested near the time the yogurt is consumed, they could metabolize the less complex oligosaccharides present, and thereby have their growth and activity stimulated. Alternatively, the probiotics (with or without other growth stimulants) can be directly added to such foods along with the appropriate combination of enzymes and prebiotics, where they can stimulate probiotic growth after consumption. 
     The combinations of enzymes and fiber sources which were shown to significantly enhance growth and activity of particular probiotics without glucose in the growth media are (note that VitaFiber™ is substantially isomalto-oligosaccharide, as shown in Table 1): 
     α-galactosidase with isomalto-oligosaccharide enhanced growth and activity of  lactobacillus  (see  FIGS. 6A ;  6 B;  FIGS. 9A ;  9 B;  10 A;  10 B;  15 A;  15 B;  16 A;  16 B;  17 A,  17 B- 20 A,  20 B); 
     β-glucanase with isomalto-oligosaccharide enhanced growth and activity of  bifidobacterium  (see  FIGS. 5A ,  5 B;  11 A;  11 B;  16 A;  16 B;  14 A;  14 B and  21 A,  21 B); 
     α-galactosidase with partially hydrolyzed guar gum enhanced growth and activity of  bifidobacterium  (see  FIGS. 7A ;  7 B); 
     β-glucanase with partially hydrolyzed guar gum enhanced growth and activity of  lactobacillus  (see  FIGS. 8A ,  8 B); 
     α-galactosidase with partially hydrolyzed guar gum enhanced growth and activity of  lactobacillus  (see  FIGS. 8A ,  8 B); 
     α-galactosidase with inulin enhanced growth and activity of  lactobacillus  (see  FIGS. 13A ,  13 B); 
     α-galactosidase with wheat dextrin enhanced growth and activity of  lactobacillus  (see  FIGS. 22A ,  22 B- 24 A,  24 B;  28 A,  28 B); 
     pectinase with wheat dextrin enhanced growth and activity of  lactobacillus  (see  FIGS. 28A ,  28 B); 
     pectinase with isomalto-oligosaccharide enhanced growth and activity of  lactobacillus  (see  FIGS. 4A ;  4 B;  9 A;  9 B;  10 A;  10 B); 
     β-glucanase with wheat dextrin enhanced growth and activity of  bifidobacterium  (see  FIGS. 25A ;  25 B- 27 A;  27 B); 
     cellulase with isomalto-oligosaccharide enhanced growth and activity of  bifidobacterium  (see  FIGS. 3A ;  3 B) 
     Tables 1, 2 and 3 below specify the fiber sources, probiotic species/strains, and the enzymes used in the Examples, which generated the results shown in the figures. 
     
       
         
               
               
               
             
           
               
                 TABLE 1 
               
               
                   
               
               
                 Fiber 
                 Brand 
                 Source 
               
               
                   
               
             
             
               
                 Partially Hydrolyzed Guar 
                 Sunfiber ® 
                 Taiyo International 
               
               
                 Gum (PHGG) 
                   
                   
               
               
                 Isomalto-oligosaccharide 
                 VitaFiber ™ 
                 BioNeutra 
               
               
                 Inulin 
                 Oliggo-Fiber ™ Instant 
                 Cargill 
               
               
                 Wheat Dextrin 
                 Benefiber ® 
                 Novartis 
               
               
                   
               
             
          
         
       
     
     
       
         
               
               
               
               
             
           
               
                   
                 TABLE 2 
               
               
                   
                   
               
               
                   
                 Probiotic 
                 Strain Designation 
                 Source 
               
               
                   
                   
               
             
             
               
                   
                 
                   Bifidobacterium lactis 
                 
                 BL-04 (BL-34) 
                 Danisco/DuPont 
               
               
                   
                 
                   Bifidobacterium lactis 
                 
                 Bi-07 
                 Danisco/DuPont 
               
               
                   
                 
                   Lactobacillus acidophilus 
                 
                 NCFM (LA-1) 
                 Danisco/DuPont 
               
               
                   
                 
                   Lactobacillus paracasei 
                 
                 LPC-37 (F-19) 
                 Danisco/DuPont 
               
               
                   
                 
                   Lactobacillus rhamnosus 
                 
                 Lr-32 (LR-44) 
                 Danisco/DuPont 
               
               
                   
                 
                   Lactobacillus plantarum 
                 
                 LP-115 (LP-29) 
                 Danisco/DuPont 
               
               
                   
                 
                   Lactobacillus salivarius 
                 
                 LS-33 (LS-30) 
                 Danisco/DuPont 
               
               
                   
                 
                   Bifidobacterium breve 
                 
                 BB-03 
                 Danisco/DuPont 
               
               
                   
                   
               
             
          
         
       
     
     
       
         
               
               
               
               
             
               
               
               
               
               
             
               
               
               
               
             
               
               
               
               
               
             
           
               
                   
                 TABLE 3 
               
               
                   
                   
               
               
                   
                 Enzyme 
                 Units 
                 Source 
               
               
                   
                   
               
             
             
               
                   
               
             
          
           
               
                   
                 Cellulase 
                 150,000 
                 CU/gm 
                 BioCat 
               
               
                   
                 Hemicellulase 
                 400,000 
                 HCU/gm 
                 BioCat 
               
               
                   
                 Pectinase 
                 3,500 
                 endo-PG/gm 
                 BioCat 
               
               
                   
                 Xylanase 
                 150,000 
                 XU/gm 
                 BioCat 
               
               
                   
                 β-Glucanase 
                 3,000 
                 BGU/gm 
                 BioCat 
               
               
                   
                 α-Galactosidase 
                 15,000 
                 GALU/gm 
                 BioCat 
               
             
          
           
               
                   
                 Fiberase (CHPX) 
                   
                 BioCat 
               
             
          
           
               
                   
                 Cellulase 
                 48,000 
                 CU/gm 
                   
               
               
                   
                 Hemicellulase  
                 102,400 
                 HCU/gm 
                   
               
               
                   
                 Pectinase 
                 420 
                 endo-PG/gm 
                   
               
               
                   
                 Xylanase 
                 25,000 
                 XU/gm 
               
               
                   
                   
               
             
          
         
       
     
     Tables 3A and 3B below explain the units used in Table 3 above. 
     
       
         
               
               
               
             
           
               
                 TABLE 3A 
               
               
                   
               
               
                 Enzyme Unit 
                   
                   
               
               
                 Abbreviation 
                 Enzymatic Unit 
                 Reference Method 
               
               
                   
               
             
             
               
                 CU 
                 Cellulase unit 
                 FCC 8 th  Edition 
               
               
                 HCU 
                 Hemicellulase unit 
                 FCC 8 th  Edition 
               
               
                 endo-PG 
                 endo-Polygalacturonase 
                 Genencor International 
               
               
                   
                 unit 
                 Procedure No. ME 400.39, 
               
               
                   
                   
                 1981 
               
               
                 XU 
                 Xylanase unit 
                 1989. Appl. Microbiol. 
               
               
                   
                   
                 Biotechno1. 30: 5-10 
               
               
                 BGU 
                 β-Glucanase unit 
                 Novozymes, EB-0338.02/01 
               
               
                 GALU 
                 α-Galactosidase unit 
                 FCC 8 th  Edition 
               
               
                   
               
             
          
         
       
     
                         TABLE 3B               Enzymatic Unit   Definition                   Cellulase unit   The amount of activity that will produce a relative           fluidity change of 1 in 5 minutes in a defined           carboxymethyl cellulose substrate under the           conditions of the assay at 40° C.       Hemicellulase   That activity that will produce a relative fluidity change       unit   of 1 over a period of 5 minutes in a locust bean gum           substrate.       Endo-   The amount of enzyme that reduces the viscosity       Polygalacturonase   of the pectin solution by 50% per minute under        unit   the conditions of the assay.       Xylanase unit   The quantity of enzyme that will liberate 1 μmol per           minute of xylose from wheat arabinoxylan under           defined conditions of temperature and pH.       β-Glucanase unit   The amount of enzyme which liberates glucose to           1 μmol glucose per minute.       α-Galactosidase   The quantity of enzyme that will liberate 1 μmol        unit   per minute of p-nitrophenol under the conditions           of the assay.                    
Preparing the Growth Media
 
     For the growth and activity determinations described below and shown in the figures, the starting media composition was MRS broth, which was modified as described below. MRS broth (including the MRS broth labeled “control-scratch” in  FIGS. 1A ,  1 B,  2 A,  2 B) consisted of the following: 
     
       
         
               
               
               
               
             
           
               
                   
                   
               
             
             
               
                   
                 Proteose Peptone #3 
                 4.0 
                 gms 
               
               
                   
                 Beef Extract 
                 4.0 
                 gms 
               
               
                   
                 Yeast Extract 
                 2.0 
                 gms 
               
               
                   
                 Glucose (or substitute ingredient) 
                 8.0 
                 gms 
               
               
                   
                 Polysorbate 80 (or LactoStim ®) 
                 0.4 
                 gms 
               
               
                   
                 Ammonium Citrate 
                 0.8 
                 gms 
               
               
                   
                 Sodium Acetate 
                 2.0 
                 gms 
               
               
                   
                 Magnesium Sulfate 
                 0.04 
                 gms 
               
               
                   
                 Manganese Sulfate 
                 0.02 
                 gms 
               
               
                   
                 Dipotassium Phosphate 
                 0.8 
                 gms 
               
               
                   
                 DI Water 
                 400 
                 mls 
               
               
                   
                   
               
             
          
         
       
     
     As noted, the starting MRS broth (by Difco™), included glucose. The glucose was removed to generate No-G-Broth, and then an equivalent quantity of one of the fiber sources in Table 1 (i.e., VitaFiber™, PHGG, Inulin or Wheat Dextrin) was added into the No-G-Broth broth, to generate each In different formulations noted in the figures and their description. In cases where an enzyme is included in the formulation in the figures and their description, the enzyme and the fiber were first reacted, then the remaining ingredients in the No-G-Broth were added (as described further below). In cases where LacoStim™ is included, following the reaction between the enzyme and fiber, the remaining ingredients in No-G-Broth were added, but not polysorbate 80. 
     To determine growth and activity with different media, enzymes and bacterial strains, the fiber source was substituted for glucose in MRS broth and filled into 500 ml flasks, which were then autoclaved at 121° C. for 15 minutes. Each flask was tempered to 37° C. and aseptically inoculated with 0.14 gram (Table 4) of one of the freeze-dried probiotic strain(s) listed in Table 2. The CFUs (colony forming units) of 0.14 gram of each strain in Table 2 is shown in the right-hand column in Table 4. 
     
       
         
               
               
               
             
           
               
                 TABLE 4 
               
               
                   
               
               
                 Probiotic species 
                 Strain 
                 CFUs from 0.14 gram 
               
               
                   
               
             
             
               
                 
                   Bifidobacterium lactis 
                 
                 BL-04 (BL-34) 
                 134.4 × 10 9   
               
               
                 
                   Bifidobacterium lactis 
                 
                 Bi-07 
                  92.4 × 10 9   
               
               
                 
                   Lactobacillus acidophilus 
                 
                 NCFM (LA-1) 
                  54.6 × 10 9   
               
               
                 
                   Lactobacillus paracasei 
                 
                 LPC-37 (F-19) 
                   42 × 10 9   
               
               
                 
                   Lactobacillus rhamnosus 
                 
                 Lr-32 (LR-44) 
                  31.5 × 10 9   
               
               
                 
                   Lactobacillus plantarum 
                 
                 LP-115 (LP-29) 
                  88.9 × 10 9   
               
               
                 
                   Lactobacillus salivarius 
                 
                 LS-33 (LS-30) 
                  88.2 × 10 9   
               
               
                 
                   Bifidobacterium breve 
                 
                 BB-03 
                   42 × 10 9   
               
               
                   
               
             
          
         
       
     
     At specific time intervals, a 30 ml sample from each flask was aseptically transferred into a HACH 2100N Turbidimeter cell. The turbidity of each sample was read and the turbidity results were reported in NTU&#39;s, where greater turbidity indicates greater growth. The same 30 ml sample that was used for the turbidity reading was transferred into a 250 ml glass beaker, and the pH was recorded. The sample was then titrated using 0.1N NaOH to an end point of pH 6.8, and the quantity of NaOH used was recorded. The % Lactic Acid was calculated using the following formula: 
     
       
         
           
             
               
                 
                   
                     % 
                     ⁢ 
                     
                         
                     
                     ⁢ 
                     Lactic 
                     ⁢ 
                     
                         
                     
                     ⁢ 
                     Acid 
                   
                   = 
                   
                     
                       
                         
                           
                             
                               Lactic 
                               ⁢ 
                               
                                 
                                     
                                 
                                 ⁢ 
                                 
                                     
                                 
                               
                               ⁢ 
                               Acid 
                             
                             = 
                             
                               ( 
                               
                                 
                                   ( 
                                   
                                     mls 
                                     ⁢ 
                                     
                                         
                                     
                                     ⁢ 
                                     of 
                                     ⁢ 
                                     
                                         
                                     
                                     ⁢ 
                                     0.1 
                                     ⁢ 
                                     N 
                                     ⁢ 
                                     
                                         
                                     
                                     ⁢ 
                                     
                                       Na 
                                       ⁢ 
                                       OH 
                                     
                                   
                                   ) 
                                 
                                 × 
                               
                             
                           
                         
                       
                       
                         
                           
                             
                               
                                 
                                   ( 
                                   
                                     90 
                                     ⁢ 
                                     
                                         
                                     
                                     ⁢ 
                                     gm 
                                     ⁢ 
                                     
                                       / 
                                     
                                     ⁢ 
                                     mole 
                                   
                                   ) 
                                 
                                 × 
                                 
                                   ( 
                                   
                                     1 
                                     ⁢ 
                                     
                                         
                                     
                                     ⁢ 
                                     L 
                                     ⁢ 
                                     
                                       / 
                                     
                                     ⁢ 
                                     1000 
                                     ⁢ 
                                     
                                         
                                     
                                     ⁢ 
                                     ml 
                                   
                                   ) 
                                 
                               
                               ) 
                             
                             × 
                             100 
                           
                         
                       
                     
                     
                       mls 
                       ⁢ 
                       
                         
                             
                         
                         ⁢ 
                         
                             
                         
                       
                       ⁢ 
                       of 
                       ⁢ 
                       
                           
                       
                       ⁢ 
                       sample 
                     
                   
                 
               
             
             
               
                 
                   
                     % 
                     ⁢ 
                     
                         
                     
                     ⁢ 
                     Lactic 
                     ⁢ 
                     
                         
                     
                     ⁢ 
                     Acid 
                   
                   = 
                   
                     
                       
                         ( 
                         
                           mls 
                           ⁢ 
                           
                               
                           
                           ⁢ 
                           of 
                           ⁢ 
                           
                               
                           
                           ⁢ 
                           0.1 
                           ⁢ 
                           N 
                           ⁢ 
                           
                               
                           
                           ⁢ 
                           
                             Na 
                             ⁢ 
                             OH 
                           
                         
                         ) 
                       
                       × 
                       
                         ( 
                         0.9 
                         ) 
                       
                     
                     30 
                   
                 
               
             
           
         
       
     
     Higher % Lactic acid indicates higher activity. For samples that were pre-digested with enzymes, the fiber source (table 1) was added to 400 mls of de-ionized water along with the enzyme(s) and incubated for 24 hours in a 37° C. water bath. The remaining MRS components (as specified in the figures and their description) were then added to each flask and autoclaved at 121° C. for 15 minutes. Each flask was tempered to 37° C. and aseptically inoculated with 0.14 gram of the specified probiotic strain(s). At specific time intervals, 30 ml samples were aseptically taken and the turbidity, pH and % Lactic Acid was determined for each flask as described above. All probiotics were held at −10° F. prior to use. All enzymes were held at 5° C. prior to use. 
     EXAMPLE#1 
     Isomalto-oligosaccharide prebiotic (VitaFiber™) was substituted as the carbohydrate source in MRS broth, replacing glucose. Growth of  Bifidobacterium lactis  (BL-04) and  Lactobacillus acidophilus  (NCFM) were monitored. BL-04 grew better with the isomalto-oligosaccharide than with glucose ( FIG. 1A ). NCFM growth was stimulated by the isomalto-oligosaccharide, however, not as much as with the glucose control ( FIG. 2A ). 
     EXAMPLE#2 
     Isomalto-oligosaccharide (VitaFiber™) was digested for 24 hours in a 37° C. water bath with 0.2% (w/v) of various enzymes. Enzymes tested were either Fiberase™ (which is a combination of cellulase, hemicellulase, pectinase and xylanase), and cellulase, hemicellulase, pectinase and xylanase were also tested individually. Digesting VitaFiber™ (VF) with Fiberase™, cellulase, hemicellulase or xylanase gave a higher activity for BL-04 than undigested VF. Pectinase did not. The highest activity occurred with cellulase (1.368% lactic acid) ( FIG. 3B ). All flasks with enzyme digested fiber and NCFM had a higher activity than the undigested fiber control. In the case of NCFM, pectinase ( FIG. 4B ) gave the highest activity with 1.350% lactic acid produced. 
     EXAMPLE#3 
     VitaFiber™ (VF) was digested with either 0.2% (wt/vol) β-glucanase or 0.2% (wt/vol) α-galactosidase and inoculated with either BL-04 or NCFM. Undigested, unheated controls were also tested, replacing media autoclaved with VF with cold filtered VF. For NCFM, VF digested with α-galactosidase had the highest activity, producing 1.413% lactic acid ( FIG. 5B ). For BL-04, VF digested with β-glucanase had the highest activity, producing 1.473% lactic acid ( FIG. 6B ). The enzyme used to digest VitaFiber™ (VF) appears to stimulate specific bacteria. VF digested with β-glucanase stimulates BL-04, but it does not have the same effect on NCFM. VF digested with α-galactosidase stimulates NCFM, but does not have the same effect on BL-04. 
     EXAMPLE#4 
     Another prebiotic fiber source, PHGG (partially hydrolyzed guar gum), was digested with 0.2% (wt/vol) β-glucanase or 0.2% (wt/vol) α-galactosidase but showed little stimulation of either Bidfidobacterium  lactis  ( FIGS. 7A ;  7 B) or  Lactobacillus acidophilus  ( FIG. 8A ;  8 B). 
     EXAMPLE#5 
       Lactobacillus plantarum  (LP-115), was assayed in both undigested VF, VF digested with 0.2% (wt/vol) pectinase or a blend of 0.1% (wt/vol) pectinase plus 0.1% (wt/vol) α-galactosidase ( FIGS. 9A ;  9 B). The activity of VF digested with 0.1% pectinase plus 0.1% α-galatosidase had an activity of 1.656% lactic acid. The blended enzyme digestion had a higher activity than pectinase alone. 
     EXAMPLE#6 
     VitaFiber™ (VF) was digested with equal amounts (by weight) of two enzymes for each strain.  Bifidobacterium lactis  (BL-04) was inoculated into VF digested with 50:50 (wt:wt), cellulase/β-glucanase (0.2% w/vol).  Lactobacillus acidophilus  (NCFM) was inoculated into VF digested with 50:50 (wt:wt), pectinase/α-galatosidase (0.2% w/vol). LactoStim™ (0.1%) was added with the other ingredients for MRS broth (but not glucose or polysorbate 80) following digestion. Ex. 3 ( FIGS. 5A ;  5 B;  6 A;  6 B) demonstrates that β-glucanase is the preferred enzyme for growing BL-04 and that α-galactosidase is the preferred enzyme for growing NCFM. VF digested with α-galactosidase, followed by adding LactoStim™, had an activity of 1.416% lactic acid for NCFM ( FIG. 10B ). The addition of LactoStim™ thus generated a slight increase in activity, when these results are compared to the  FIG. 5B  results. VF digested with β-glucanase followed by adding LactoStim™, had an activity of 1.365% lactic acid ( FIG. 11B ) for BL-04. The addition of LactoStim™ thus generated a slight decrease in activity, as seen when these results are compared to the  FIG. 6B  results. LactoStim™ is a patented probiotic stimulant protected by U.S. Pat. Nos. 8,105,576 and 8,105,577. 
     EXAMPLE#7 
     Inulin was used as the fiber source in testing the growth and activity of  Bifidobacterium lactis  (BL-04). Inulin was digested with 0.2% (wt/vol) β-glucanase ( FIGS. 12A ;  12 B).  Lactobacillus acidophilus  (NCFM) was tested with inulin or inulin digested with 0.2% (wt/vol) α-galatosidase, both with and without addition of 0.1% (wt/vol) LactoStim™ following digestion ( FIGS. 13A ;  13 B). Digested inulin showed a small increase in activity. 
     EXAMPLE#8 
     VitaFiber™ was digested with varied amounts of either a combination of 50:50 (wt:wt) β-glucanase plus α-galactosidase, β-glucanase alone, or α-galatosidase alone. VF digested with half the amount of β-glucanase (0.1% w/vol) had an activity of 1.290% lactic acid for BL-04. This activity was less than when 0.2% (w/vol) β-glucanase was used to digest VF ( FIG. 14B ). Digesting VF with 0.05% (wt/vol) β-glucanase plus 0.05% (wt/vol) α-galactosidase had an activity of 1.362% lactic acid for NCFM, while digesting VF with 0.1% (wt/vol) β-glucanase plus 0.1% (wt/vol) α-galactosidase had an activity of 1.314% lactic acid for NCFM. Digesting VF with 0.1% (w/vol) α-galactosidase alone had an activity of 1.290% lactic acid for NCFM ( FIG. 15B ). 
     EXAMPLE#9 
     VitaFiber™ was digested with β-glucanase plus α-galactosidase at either 3:1 or 1:3 (wt:wt) ratios, both with and without subsequent addition of LactoStim™. Each flask was inoculated with 0.14 gram of a 50:50 mix (wt:wt) of BL-04 plus NCFM. The highest activity occurred when VF was digested with 1:3 (wt:wt) β-glucanase/α-galactosidase at 0.2% (wt/vol) followed by adding 0.1% (wt/vol) LactoStim™. This resulted in an activity of 1.512% lactic acid ( FIG. 16B ). This activity is higher than either of the highest digested VF tests assayed with a single bacterial strain of  Bifidobacterium lactis  or  Lactobacillus acidophilus.    
     EXAMPLE#10 
     The 1:3 ratio of β-glucanase/α-galactosidase (wt:wt) at 0.2% (wt/vol) was also tested with strains of  Lactobacillus salivarius  (LS-33),  Lactobacillus paracasei  (LPC-37),  Lactobacillus plantarum  (LP-115),  Lactobacillus rhamnosus (Lr-32), and  Bifidobacterium lactis  (Bi-07 strain). As noted with previous experiments,  Lactobacillus  strains had a higher activity when VitaFiber™ was digested with α-galactosidase rather than β-glucanase, and  Bifidobacterium  strains had a higher activity when VF was digested with β-glucanase rather than α-galactosidase. For LS-33, LPC-37, LP-115 and Lr-32, digestion with 0.2% (wt/vol) α-galactosidase had a slightly higher activity than with the 1:3 (wt:wt) β-glucanase/α-galactosidase blend at 0.2% (wt/vol). ( FIGS. 17B ,  18 B,  19 B,  20 B and  21 B). 
     EXAMPLE#11 
     Growth and activity of  Lactobacillus rhamnosus (Lr-32),  Lactobacillus salivarius  (LS-33) and  Lactobacillus acidophilus  (NCFM) was tested with wheat dextrin or wheat dextrin digested with α-galactosidase at 0.2% (wt./vol.). ( FIGS. 22B ;  23 B;  24 B). In all cases, the digested wheat dextrin generated a higher activity (% lactic acid). 
     EXAMPLE#12 
     Growth and activity of  Bifidobacterium lactis  (BL-04),  Bifidobacterium lactis  (Bi-07) and  Bifidobacterium breve  (BB-03) was tested with wheat dextrin or wheat dextrin digested with β-glucanase at 0.2%(wt/vol). In all cases, the enzyme digested wheat dextrin generated a higher activity ( FIGS. 25B ,  26 B;  27 B). 
     EXAMPLE#13 
     Growth and activity of  Lactobacillus plantarum  (LP-115) was tested with wheat dextrin or wheat dextrin digested with either 0.2% (wt/vol) α-galactosidase or 0.2% (wt/vol) pectinase. Wheat dextrin digested with 0.2% (wt/vol) α-galactosidase generated a higher activity ( FIG. 28B ). 
     The specific methods and compositions described herein are representative of preferred embodiments and are exemplary and not intended as limitations on the scope of the invention. Other objects, aspects, and embodiments will occur to those skilled in the art upon consideration of this specification, and are encompassed within the spirit of the invention as defined by the scope of the claims. It will be readily apparent to one skilled in the art that varying substitutions and modifications may be made to the invention disclosed herein without departing from the scope and spirit of the invention. The invention illustratively described herein suitably may be practiced in the absence of any element or elements, or limitation or limitations, which is not specifically disclosed herein as essential. Thus, for example, in each instance herein, in embodiments or examples of the present invention, any of the terms “comprising”, “including”, containing”, etc. are to be read expansively and without limitation. The methods and processes illustratively described herein suitably may be practiced in differing orders of steps, and that they are not necessarily restricted to the orders of steps indicated herein or in the claims. It is also noted that as used herein and in the appended claims, the singular forms “a,” “an,” and “the” include plural reference, and the plural include singular forms, unless the context clearly dictates otherwise. Under no circumstances may the patent be interpreted to be limited to the specific examples or embodiments or methods specifically disclosed herein. Under no circumstances may the patent be interpreted to be limited by any statement made by any Examiner or any other official or employee of the Patent and Trademark Office unless such statement is specifically and without qualification or reservation expressly adopted in a responsive writing by Applicants. 
     The invention has been described broadly and generically herein. Each of the narrower species and subgeneric groupings falling within the generic disclosure also form part of the invention. The terms and expressions that have been employed are used as terms of description and not of limitation, and there is no intent in the use of such terms and expressions to exclude any equivalent of the features shown and described or portions thereof, but it is recognized that various modifications are possible within the scope of the invention as claimed. Thus, it will be understood that although the present invention has been specifically disclosed by preferred embodiments and optional features, modification and variation of the concepts herein disclosed may be resorted to by those skilled in the art, and that such modifications and variations are considered to be within the scope of this invention as defined by the appended claims.