Chemistry: molecular biology and microbiology – Enzyme – proenzyme; compositions thereof; process for... – Hydrolase
Reexamination Certificate
2000-04-04
2004-12-21
Rao, Manjunath N. (Department: 1652)
Chemistry: molecular biology and microbiology
Enzyme , proenzyme; compositions thereof; process for...
Hydrolase
C435S004000, C435S006120, C435S014000, C435S041000, C435S091530, C435S099000, C435S105000, C435S471000, C435S252300, C435S320100, C435S173100, C435S243000, C435S252310, C435S262000, C435S252900, C426S056000, C426S522000, C536S023200, C536S023700
Reexamination Certificate
active
06833260
ABSTRACT:
FIELD OF THE INVENTION
The present invention relates to the use of lactic acid bacteria to reduce lactose in dairy products and their use in food and other commercial applications.
BACKGROUND OF THE INVENTION
Lactose, or milk sugar, is a disaccharide carbohydrate that is hydrolyzed to glucose and galactose by the enzyme lactase, also known as beta-galactosidase (&bgr;-galactosidase). Although lactase is normally present in humans in intestinal epithelial cells and thereby contributes to the digestion of lactose present in dairy foods, a significant number of the adult human population is unable to digest lactose due to a genetic deficiency of this enzyme. Persons deficient in lactase may react to the consumption of dairy products with symptoms including nausea, cramps, gas, and diarrhea, a clinical condition known as lactose intolerance. Temporary lactase deficiencies in individuals with normal lactase levels may also result from damage to the intestinal lining produced by viral or bacterial infections, cancer chemotherapy, allergic or autoimmune conditions, and from decreases in lactase associated with aging. Persons who are lactase deficient and wish to consume normal dairy products with high lactose levels such as milk (12-13 grams of lactose per glass) can either purchase lactase treated dairy foods (70-100% hydrolyzed lactose) or consume a dietary supplement of lactase enzyme before each dairy meal. The cost and properties of current lactase preparations have limited the availability of lactose-reduced or free dairy products and other foods. Improvements in enzyme production costs and hydrolysis reaction properties could lead to the availability of a larger number of lactose reduced and free dairy foods and ingredients as well as more effective prophylactic products.
SUMMARY
The invention is based, in part, on the discovery of a method of achieving enhanced lactose hydrolysis using lactic acid bacteria. More specifically, the invention relates to a method of hydrolyzing lactose by using lactic acid bacteria that produce high levels of &bgr;-galactosidase, permeablizing the bacteria such that lactose can enter the cell and be hydrolyzed by the highly concentrated &bgr;-galactosidase contained therein. The above method results in the generation of permeablized lactic acid bacteria which contain high concentrations of &bgr;-galactosidase (referred to herein as lactase microcarriers) and which have a surprisingly high ability to rapidly and efficiently hydrolyze lactose under a variety of conditions. Moreover, the lactase microcarriers also have protease and bile resistant properties making them an ideal supplemental enzyme and oral prophylactic against the clinical condition of lactose intolerance.
Accordingly, the invention features a method for preparing a lactase microcarrier for hydrolyzing lactose in a liquid, e.g., milk, a whey product, or derivatives thereof. The method includes transforming a food-grade lactic acid bacterium with a DNA construct, wherein the DNA construct includes a promoter sequence of a gene operatively linked to a DNA sequence encoding a &bgr;-galactosidase, culturing the transformed bacterium under conditions that enable expression of the &bgr;-galactosidase such that the &bgr;-galactosidase activity exhibited in the bacterium is at least about 4000 MU (e.g., at least 4500, 5000, 6000, 7000, 8000, 9000, 10,000, 11,000 or 15,000 MU), and permeabilizing the bacterium. The method can further include contacting the permeabilized bacterium with a liquid containing lactose for a time sufficient to hydrolyze the lactose. The lactic acid bacterium can be a Streptococcus, Aerococcus, Carnobacterium, Enteroccus, Erysipelothrix, Gemella, Globicatella, Lactobacillus, Lactococcus, Bidobacteria, Leuconostoccocus, Pediococcus, Streptococcus, Tetragenococcus, or Bagococcus bacteria. In particular, the lactic acid bacterium can be a
Lactococcus lactis.
The DNA sequence encoding the &bgr;-galactosidase can be selected from the group
Streptococcus thermophilus, Lactobacillus bulgaricus, Bifobacterium species, Aspergillus niger, Aspergillus oryzae, Kluyveromyces fragilis, Kluyveromyces lactis, Bacillus subtillus
or Arthrobacter species. The promoter can be a promoter from a gene that encodes an antimicrobial peptide, e.g., a lantibiotic, e.g., a nisin gene promoter, such as a nisA promoter. The bacterium can be permeabilized by an agent such as a chemical, a solvent (e.g., ethanol or isopropanol), or a detergent, (e.g., deoxycholate, sodium dodecyl sulfate, rhamnolipid, or chenodeoxycholate). In one embodiment of the method, hydrolysis of lactose is performed at 4° C. In this embodiment, at least 90% of the lactose is hydrolyzed within 6 hours by a concentration of enzyme which is equivalent to 5000 o-nitrophenyl-&bgr;-galactosidase (ONPG) units/liter. In another embodiment of the method, hydrolysis of lactose is performed at 55° C. (or up to 63° C). In this embodiment, at least 90% of the lactose is hydrolyzed within 2 hours by a concentration of enzyme equivalent to 5000 ONPG units/liter.
In another aspect, the invention features a method for hydrolyzing lactose including providing a permeabilized lactic acid bacterium containing a &bgr;-galactosidase, wherein the bacterium exhibits a &bgr;-galactosidase activity of at least about 4000 Miller units, and contacting the permeabilized bacterium with a liquid containing lactose, for a time sufficient to hydrolyze the lactose. The lactic acid bacterium can be Streptococcus, Aerococcus, Carnobacterium, Enteroccus, Erysipelothrix, Gemella, Globicatella, Lactobacillus, Lactococcus, Bidobacteria, Leuconostoccocus, Pediococcus, Streptococcus, Tetragenococcus, or Bagococcus. In particular, the lactic acid bacterium is a
Lactococcus lactis.
The &bgr;-galactosidase can be encoded by a heterologous gene, e.g., a
Streptococcus thermophilus
&bgr;-galactosidase. In other embodiments, the bacterium exhibits a &bgr;-galactosidase activity of at least about 10,000 Miller Units.
In addition, the invention also features a permeabilized lactic acid bacterium containing a heterologous &bgr;-galactosidase, e.g., a
Streptococcus thermophilus
&bgr;-galactosidase, wherein the &bgr;-galactosidase exhibits an activity of at least about 4000 Miller Units. The permeabilized bacterium can be Streptococcus, Aerococcus, Carnobacterium, Enteroccus, Erysipelothrix, Gemella, Globicatella, Lactobacillus, Lactococcus, Bidobacteria, Leuconostoccocus, Pediococcus, Streptococcus, Tetragenococcus, or Bagococcus. In particular, the lactic acid bacterium can be a
Lactococcus lactis.
The permeabilized bacterium can be in a lyophilized form, in a concentrated cell suspension, or immobilized. In one embodiment, the invention includes a composition including the permeabilized bacterium containing a heterologous &bgr;-galactosidase which exhibits at least about 4000 Miller Units. In another embodiment, the invention includes a food product for use with a dairy product, wherein the food product includes the permeabilized bacterium containing a heterologous &bgr;-galactosidase which exhibits an activity of at least about 4000 Miller Units.
Also within the invention is a method of administering, e.g., orally, lactase to a mammal, the method including administering to the mammal the permeabilized bacterium containing a heterologous &bgr;-galactosidase which exhibits at least 4000 Miller Units.
The invention further features a reduced lactose dairy product including a dairy product, e.g., milk, and a permeablized
Lactococcus lactis.
The
Lactococcus lactis
can contain a
Streptococcus thermophilus
&bgr;-galactosidase.
“Food-grade bacteria” are microorganisms that are routinely consumed either as ingredients in fermented foods (e.g., cheese, bread, beer, yogurt) or as food or dietary supplements which aid in digestive processes (e.g. Lactobacilli, Bifidobacteria) and which have a record of being safe and non-toxic to consumers.
An “isolated nucleic acid” is a nucleic acid which has a non-naturally occurring sequence, or which has the sequence of part or
Fish & Richardson P.C.
Protein Scientific, Inc.
Rao Manjunath N.
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