DK143714B - METHOD FOR HYDROLYSE OF LACTOSE UNDER GLUCOSE AND GALACTOSE CREATION - Google Patents

Description

09) DENMARK (^ |)

| p (12) PREFACE SCRIPTURE (ni 143714B

DIRECTORATE OF THE PATENT AND TRADEMARKET SYSTEM

(21) Application No. 5 ^ 51/75 (51) lnt.CI.3 C 12 P 19/14 (22) Filing Day 3 · Dec. 1975 (24) Race day 5 · dec. 1975 (41) Aim. available 5 Jun. 1976 (44) Presented 28 Sep. 1981 (86) International application no.

(86) International filing day (85) Continuation day - (62) Master application no. -

(30) Priority 4 Dec. 1974, 529329, US

(71) Applicant R. J. REYNOLDS TOBACCO COMPANY, Winston-Salem, US.

(72) Inventor Margaret E. Long, US: Chin K. Lee, US.

(74) Associate Engineer Hofman-Bang & Bout ard.

(54) Process for hydrolysis of lactose to form glu »= cose and galactose.

The present invention relates to a process for hydrolysis of lactose to form glucose and galactose, and the process of the invention is characterized by contacting the lactose with a lactase produced by cultivation of the organism Bacillus coagulans NRRL B-8100 in a lactose-containing nutrient medium.

q In recent years, there has been considerable interest in pursuit. develop methods for reducing the lactose content of milk and “products derived from milk. This interest has been heightened by recent evidence that a large percentage of the human population suffers from lactase deficiency, which is either an inherited property or a result of "an aging process.

t 2 143714 Such lactase deficiency leads to intestinal tract disorders where the lactose content of the food is high. Furthermore, similar lactose intolerance has been observed in some domestic animals.

The hydrolysis of lactose in milk and milk-derived products to form glucose and galactose is an attractive target not only because it would solve the lactose intolerance problem, but also because it would increase the sweetness of the products and reduce the so-called sandy textures in certain milk-derived products, caused of lactose crystallization. Those skilled in the art have long recognized the desirability of inducing this hydrolysis using lactase. Despite the fact that lactase is relatively widespread in nature and produced by many microorganisms, the use of lactase in the commercial production of milk and milk-derived products has been very limited. One of the reasons for the limited commercial use of lactase is that many of the common lactases, such as yeast derived lactases, exhibit optimal enzyme activity at temperatures also leading to bacterial growth. As a result, there has been a growing interest in finding lactase with a high degree of thermostability. Such thermostable lactase would allow lactose hydrolysis to be carried out under conditions which are unfavorable to the growth of certain bacteria frequently present in milk or milk derived products. Such a lactase derived from Streptomyces coelicolor is described in U.S. Patent No. 3,816,259.

It is not clear at this time whether S. coelicolor lactase can be used without risk because some members of this species have been shown to produce antibiotics.

The production of lactase by members of the Bacillus genus has been described previously. Thus, P.J. Anema, Biochem. Biophys.

Acta, §2 (3), 495-502 (1964) described the isolation of lactase from B. subtilis. B. megaterium lactase is described by S.R. Rohlfing and I.P. Crawford, J. Bacteriology 92 (4), 1258-9 (1966). However, none of these organisms can be considered heat-tolerant, and the lactase formed must generally be used at temperatures below 50 ° C to maintain useful enzyme activity for an extended period of time.

It is the object of the present invention to provide a process for the hydrolysis of lactose to form glucose and galactose at relatively high temperatures of up to 70 ° C, especially between 45 and 65 ° C, and this is achieved by toxin in contact with a lactase produced by the cultivation of a novel organism of the genus Bacillus with particularly advantageous properties. Thus, it exhibits optimum growth at a temperature of at least approx. 45 ^ 0th The organism, designated Bacillus coagulans NRRL B-8100, is grown in a nutrient medium containing lactose and then the lactase formed is isolated.

The organism used for this purpose is isolated from a soil sample. Cultivation of the organism in a suitable nutrient medium containing lactose leads to the formation of the desired lactase intracellularly. A characteristic of the culture has been made and it has been identified as declining within the species Bacillus coagulans according to the classification according to Bergey's Manual of Determinative Bacteriology, 7th Edition. This organism is deposited in the cultural collection of the U.S.D.A. Northern Regional Research Laboratory under the designation NRRL B-8100. The taxonomic properties of this strain are described in Table 1 below.

TABLE 1 A. Morphological characteristics: 1. Vegetative rods: Less than 0.9 µm in diameter, length varying in general up to 5.0 - 6.0 µιη. Some filaments. No chains. Gram positive, uniform staining. Movable.

2. Sporangia: As a rule, not swollen, but some swollen bronchi can be found.

3. Spores: 0.9 x 1.2 - 1.5 µm, ellipsoidal, subterminal Until terminal.

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B. Cultural characteristics: 1. Gelatin agar No hydrolysis, line culture: 2. Agar colonies: Opaque, small, round.

Not distinguishable.

3. Agar slant cultures: Scattered to moderate growth. Flat, smooth, transparent.

4. Glucose agar Growth more powerful than nutritional culture: agar. Smooth, white.

5. Glucose asparagine No growth for 24 hours, agar culture: Moderate growth after 48 hours.

6. Acid Proteosis- Good growth, better than on nutrient peptone agar goblet.

cultures: 7. Soybean agar- Moderate growth, slightly stronger than oblique cultures: on nutrient agar.

8. Slant culture on Scattered growth after 24 hours, agar for living as good as nutritional agar for lasting: 48 hours.

9. Meat Soup: Poor growth after 24 hours.

10. Sodium Chloride- No growth in 7% sodium chloride. meat soup: 11. Milk agar line- No hydrolysis, culture: 12. Potato: Scattered, dry, wrinkled.

C. Physiological characteristics: 1. Using peptone as a nitrogen source, the organism produced acid but no gases from glucose, lactose, arabinose, xylose, mannitol and maltose.

Neutral reaction to sucrose and glycerol.

2. The pH of glucose nutrient fluid is 5.0 or less in 7 days.

3. No citrate utilization.

4. Tomato yeast milk ran out after 24 hours at 45 ° C, 5. No production of nitrates from nitrates.

6. Negative Voges-Proskauer test. The pH of the Voges-Proskauer liquid was 4.2.

7 · Positive starch hydrolysis.

Positive catalase reaction.

9. No washing on nitrate-containing medium under anaerobic conditions. Growth on glucose nutrient fluid under anaerobic conditions leads to pH below 5.2 over 7 days.

10. Aerobic, optional anaerobic, 11. Minimum temperature for growth is 25 ° C, Maximum temperature for growth is 60 ° G. Optimal growth at 45 - 50 ° C.

As indicated under the physiological characteristics of Table 1, the described Baeillus organism exhibits optimum growth at temperatures of about 45 - 50 ° C and can therefore be perceived as a heat tolerant organism in comparison to other Bacillus * o species exhibiting optimum growth at ca. 37 C. A heat-tolerant Baeillus organism is defined herein as an organism that exhibits optimum growth at temperatures of about 45 ° C and above.

The invention is further illustrated by the following example.

EXAMPLE

The pH optimum for the lactase produced by the organism was determined by analyzing whole cells in the presence of o-nitrophenyl-β-galactoside (ONPG) as a substrate. It proceeded essentially as described by J. Lederberg, J. Bacteriology 60, 381 (1950). The cells were first treated with toluene using a phosphate buffer and the assay temperature was 37 ° C.

The stability of the lactase produced by B, coagulans was assessed by using ONPG as a substrate in a modified Leder-Berg method. For this study, washed out cells were suspended in 0.05 M phosphate buffer at pH 7.0 in the presence of the ONPG substrate. The suspension was kept at 60 ° C for four days, taking samples periodically to determine residual lactase activity. The temperature of this stability test was chosen as approximately the temperature levels used in low temperature pasteurization. The results of the tests are shown in Table 2.

TABLE 2

Time (hours) Lactase activity as pot.

of initial activity 0 100 21 92 45 51 71 36 99 20

The lactase produced by Bacillus coagulans exhibits useful enzyme activity up to 70 ° C. Optimal activity appears to occur at temperatures of 60-65 ° C. The particular temperature selected for carrying out lactose hydrolysis using this enzyme will depend on some of the enzyme used. substratmedium. Generally, however, temperatures of 45 - 65 ° C are preferred.

A typical medium for growing Bacillus coagulans during lactase production has the following composition:

Proteose-peptone .......................... 1.0% Yeast Extract ................ i ............. 1.0%

Potassium dihydrogen phosphate ................. 0.8%

Lactose (sterilized separately) ........... 2.0% pH ............................. .......... 6.0

Cultures are incubated on a rotary shaker for 48 hours at 45 ° C. After completion of the incubation period, toluene is added.

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to the liquid (0.5% by volume) and the mixture is stirred for 30 minutes. The cells are then recovered by flocculation, as described in U.S. Patent No. 3 * 821,086, and the resulting flocculated cell aggregate is dried at 55 ° C. The lactase activity of the dried aggregate particles is 38.5 units per minute. grams, where one unit is defined as the amount of enzyme needed to generate one micromole of dextrose per gram. minute under the test conditions. The test method used is described by Weetall et al., Biotechnology and Bioengineering 16, 295 (1974).

The efficacy of the lactase produced by Bacillus coagulans was demonstrated by the hydrolysis of lactose in a sweet stored whey. The organism was cultured and the cells were watered as described above. The dried aggregate particles were sieved and 5 g of the 16-20 mesh portion (0.017-0.024 µm) hydrated in a 50% lactose solution buffered to pH 7.0 with a 0.05 phosphate buffer. The hydrated particles were then placed in a small glass column maintained at a temperature of 60 ° C. Through this packed column, an aqueous solution containing 70 grams of a commercially dried sweet whey powder was continuously passed. The solution is buffered with 0.05 M phosphate to pH 7.0. The solution contained about 5% by weight of lactose, calculated on the lactose content of the sweet whey, and 100 mg / liter of methyl p-hydroxybenzoate was added as a preservative. The flow rate through the column was maintained at 375 ml / day and the degree of lactose hydrolysis was determined daily by routine analysis. The initial rate of lactose hydrolysis was found to be 90 per cent. After 3 weeks of continuous operation, the degree of hydrolysis had dropped to 80 per cent.

It will be appreciated that the lactase produced can be used either for batch or continuous treatment with lactose-containing substrates. Furthermore, the lactase can be used by direct use of the cells or it can be used in the form of the cell-free enzyme by methods known per se.