CN102206599A - Oxygen-resistant acid-resistant Bifidobacterium longum - Google Patents

Abstract

The invention relates to an oxygen-resistant and acid-resistant Bifidobacterium longum, characterized in that: the name is CC-1, the classification name is Bifidobacterium longum, the preservation number is: CGMCC No.4730, the preservation date is April 2, 2011, and the preservation address For: No. 3, Yard No. 1, Beichen West Road, Chaoyang District, Beijing. Preservation unit: General Microbiology Center of China Committee for the Collection of Microbial Cultures. The invention effectively improves the physiological and biochemical properties of bifidobacteria, enhances the tolerance of bifidobacteria to oxygen and acid, and puts oxygen-resistant and acid-resistant Bifidobacterium longum in a PTYG liquid medium with a pH value of 4.0 under normal aerobic conditions. Cultivated at 37°C, the number of viable bacteria of oxygen-resistant and acid-resistant Bifidobacterium longum CC-1 can reach 4.7×10 ⁷ CFU/ml from the counting results of viable bacteria by the pouring method. has an extremely important role.

Description

A kind of oxygen-resistant and acid-resistant Bifidobacterium longum

technical field

The invention relates to the technical field of food science, in particular to an oxygen-resistant and acid-resistant Bifidobacterium longum.

Background technique

In 2002, FAO/WHO defined probiotics as "live microorganisms that have health effects on the host after taking sufficient quantities". Bacteria as probiotics should meet the following conditions: (1) beneficial to the host; (2) non-toxic and non-pathogenic; (3) able to survive in the digestive tract; (4) able to adapt to gastric acid and bile salts; (5) ) can colonize the surface of the digestive tract; (6) can produce useful enzymes and metabolites; (7) can maintain activity during processing and storage; (8) have good sensory properties. On May 29, 2005, China's State Food and Drug Administration promulgated the "Regulations on the Declaration and Review of Probiotic Health Foods", and announced a batch of probiotic strains that can be used in health foods, including: Bifidobacterium bifidum bifidum), Bifidobacterium infantis, Bifidobacterium longum, Bifidobacterium breve, Bifidobacterium adolescentis, Lactobacillus delbrueckii subsp. bulgaricus), Lactobacillus acidophilus, Lactobacillus casei subsp. Casei, Streptococcus thermophilus, Lactobacillus reuteri. At present, the probiotics used to make functional foods are mainly Lactobacillus and Bifidobacterium. Different types of probiotics have different effects. In comparison, Bifidobacteria are stronger in regulating the function of the gastrointestinal tract.

Bifidobacteria widely exist in the intestines of humans and warm-bodied animals, the female reproductive tract, and the rumen of ruminants. It occupies a high proportion in the intestines of infants and adults. main microorganisms. As early as 1899, Henry Tissier of the Pasteur Institute in France first isolated Bifidobacteria from the feces of breastfed infants. The focus of bacterial research. The health functions of bifidobacteria include: (1) inhibit pathogenic bacteria, prevent diarrhea and constipation, and have inhibitory effects on spoilage bacteria, pathogenic Escherichia coli, Staphylococcus aureus, Shigella dysenteriae, Proteus, etc.; (2) Prevention and treatment of hypertension and arteriosclerosis, bifidobacteria can convert cholesterol into steroids that are not absorbed by the human body, reduce the concentration of cholesterol in the blood, and have a certain preventive effect on hypertension and arteriosclerosis, bifidobacteria can also reduce serum cholesterol and glycerin Diesters have the effect of improving lipid metabolism disorders; (3) nutrition, bifidobacteria can synthesize a variety of vitamins, such as VB ₁ , VB ₂ , VB ₆ , VB ₁₂ , folic acid, nicotine, pantothenic acid, etc., metabolize The organic acid produced can reduce the pH value and Eb value in the environment, which is beneficial to the absorption of ferrous iron, calcium and vitamin D; (4) immune and anti-aging effects, activate the immune system of the body, so that it can maintain immune surveillance and immune clearance functions , constantly remove aging, dead cells and mutant cells, so that the body will not age due to dead cells and waste accumulation; Bifidobacteria can increase the activity and content of superoxide dismutase in the blood, thereby reducing the oxidation reaction involving free radicals (5) Reduce the production and absorption of intestinal-derived endotoxins. Bifidobacteria can absorb and utilize nitrogen-containing harmful substances in the intestines, inhibit spoilage bacteria that produce amines, reduce the pH value in the intestines, and make ammonia change. It is an ion type that is difficult to absorb, so as to achieve the effect of reducing blood ammonia, thereby reducing the harm to the liver; (6) inhibiting the effect of tumors, bifidobacteria can activate macrophages in the immune system of the body, enhance immune function, reduce The level of cancer mutagens in the body, which inactivates the enzymes that produce cancer mutagens. In addition, intestinal saprophytic bacteria can produce many amine carcinogens in their metabolism, and some can convert some carcinogenic precursors into carcinogens. Bifidobacteria prevent intestinal (7) Improving and regulating intestinal flora imbalance, flora imbalance (a large reduction of bifidobacteria) can lead to intestinal dysfunction, children can cause persistent and recurrent infections, high levels of suitable bifidobacteria Passing in the intestinal tract can prevent the pathogenic effect of pathogens, so that the diarrhea caused by intestinal flora imbalance can be corrected.

Of course, the bifidobacteria in the human body must reach a certain amount before they can exert their beneficial effects. At present, it is generally believed that the amount of viable bifidobacteria in each gram of intestinal contents must reach at least 1×10 ⁷ Its specific parts of the gut work. There are many factors that affect the final survival rate of bifidobacteria, including the strain itself, oxygen partial pressure, pH value, bile salt content, etc. Bifidobacteria are obligate anaerobic bacteria and are not acid-resistant. Under these conditions, the growth is slow and easy to die, which makes the cultivation difficult and the production cost is high, which brings great difficulties to the manufacturers of bifidobacterium products.

Contents of the invention

The purpose of the present invention is to overcome the deficiencies of the prior art, by carrying out continuous oxygen-resistant and acid-resistant acclimatization to Bifidobacterium longum, obtain oxygen-resistant and acid-resistant Bifidobacterium longum CC-1, the number of viable bacteria after the culture of this strain It can reach 4.7×10 ⁷ CFU/mL.

The purpose of the present invention is achieved through the following technical solutions:

An oxygen-resistant and acid-resistant Bifidobacterium longum, the name is CC-1, the classification name is Bifidobacterium longum, the preservation number is: CGMCC No.4730, the preservation date is April 2, 2011, and the preservation address is: Beichen, Chaoyang District, Beijing No. 3, No. 1 Yard, West Road, preservation unit: General Microbiology Center of China Microbiological Culture Collection Management Committee.

Moreover, the number of viable bacteria of the oxygen-resistant and acid-resistant Bifidobacterium longum CC-1 is 4.7×10 ⁷ CFU/mL.

Advantage of the present invention and positive effect are as follows:

The invention effectively improves the physiological and biochemical properties of bifidobacteria, enhances the tolerance of bifidobacteria to oxygen and acid, and puts oxygen-resistant and acid-resistant Bifidobacterium longum in a PTYG liquid medium with a pH value of 4.0 under normal aerobic conditions. Cultured at 37°C, according to the counting results of viable bacteria by the pouring method, the number of viable bacteria of oxygen-resistant and acid-resistant Bifidobacterium longum CC-1 can reach 4.7×10 ⁷ CFU/mL. has an extremely important role.

Description of drawings

Fig. 1 is the change curve of the OD ₆₀₀ value of the thalline after the oxygen-resistant and acid-resistant Bifidobacterium longum CC-1 of the present invention and the initial Bifidobacterium longum are cultivated in PTYG liquid medium with a pH value of 7.0 under anaerobic conditions at 37°C .

Fig. 2 is the change curve of the bacterial cell pH value after the oxygen-resistant and acid-resistant Bifidobacterium longum CC-1 and initial Bifidobacterium longum of the present invention are cultured in PTYG liquid medium with a pH value of 7.0 at 37° C. under anaerobic conditions.

Detailed ways

Below in conjunction with the examples, the present invention is further described, the following examples are illustrative, not limiting, and the protection scope of the present invention cannot be limited by the following examples.

1. Acclimatization of Bifidobacterium longum

Bifidobacterium basal medium PTYG ingredients: soybean peptone 5g, tryptone 5g, yeast extract 10g, glucose 10g, L-cysteine hydrochloride 0.5g, trace salt solution 40mL, water 1000mL, adjust the pH value to 7.0. The composition of trace salt solution is (1000mL water): CaCl ₂ : 0.2g, MgSO ₄ 7H ₂ O: 0.48g, KH ₂ PO ₄ : 1.0g, NaHCO ₃ : 10.0g, K ₂ HPO ₄ : 1.0g, NaCl: 2.0g.

The initial strain was Bifidobacterium longum existing in the laboratory.

Inoculum size: 5%, culture temperature: 37°C

(1) Oxygen-resistant acclimatization of Bifidobacterium longum: Acclimate by gradually increasing the oxygen partial pressure in the medium, subculture 5-8 times under each oxygen partial pressure gradient and repeatedly acclimatize, and then inoculate to the next oxygen partial pressure Gradient cultivation, and after each oxygen-resistant acclimatization, a single colony of aerobic-resistant Bifidobacterium longum was selected.

The present invention increases the partial pressure of oxygen in the culture medium through the following approaches: the one is to cultivate in an anaerobic box, increase the partial pressure of oxygen by 0.02 each time, and finally transfer to common culture conditions for cultivation; the other is to change the cultivation environment At the same time, under certain conditions of the culture vessel, gradually reduce the liquid volume, that is, use a 100mL anaerobic bottle, and the liquid volume is 100mL, 80mL, 60mL, 40mL, and 20mL.

(2) Acid-resistant acclimation of Bifidobacterium longum: The oxygen-resistant Bifidobacterium longum obtained after the oxygen-resistant acclimation is then subjected to acid-resistant acclimation, by gradually reducing the pH value of the medium for acclimation, and the pH values are 7.0, 6.5, 6.0, 5.5, 5.0, 4.5, 4.0, each pH value gradient was subcultured 5-8 times, repeatedly acclimated, and then inoculated to the next pH value, and selected acid-resistant Bifidobacterium longum single colonies after each acid-resistant acclimation, and finally obtained the best growth ability. Strong CC-1.

2. Growth characteristics of oxygen-resistant and acid-resistant Bifidobacterium longum

The growth characteristics of oxygen-resistant and acid-resistant Bifidobacterium longum can be known by analyzing and comparing the OD ₆₀₀ value, pH value, and the number of viable bacteria of the initial bacteria and the growth of oxygen-resistant and acid-resistant Bifidobacterium longum.

It can be seen from the change curves of the OD ₆₀₀ value and pH value of the bacteria after the oxygen-resistant and acid-resistant Bifidobacterium longum CC-1 and the initial bacteria are simultaneously cultured in the PTYG liquid medium with a pH value of 7.0 at 37°C under anaerobic conditions, The oxygen-resistant and acid-resistant strains grow better, and the lag period is slightly shortened, the number of viable bacteria is relatively high, and the acid-producing ability is also improved.

The oxygen-resistant and acid-resistant Bifidobacterium longum CC-1 and the initial bacteria were simultaneously cultured in the PTYG liquid medium with a pH value of 4.0 under normal aerobic conditions at 37°C. According to the counting results of viable bacteria by the pouring method, the oxygen-resistant and acid-resistant Bifidobacterium longum The viable count of bacilli can reach 4.7×107CFU/mL, while the initial strain can only reach 2.1×103CFU/mL.

Claims (2)

1. An oxygen-resistant and acid-resistant Bifidobacterium longum, characterized in that: the name is CC-1, the classification name is Bifidobacterium longum, the preservation number is: CGMCC No.4730, the preservation date is April 2, 2011, and the preservation address is : No. 3, Yard No. 1, Beichen West Road, Chaoyang District, Beijing. Preservation unit: General Microorganism Center of China Committee for the Collection of Microbial Cultures. 2. The oxygen-resistant and acid-resistant Bifidobacterium longum according to claim 1, characterized in that: the number of viable bacteria of the oxygen-resistant and acid-resistant Bifidobacterium longum CC-1 is 4.7×10 ⁷ CFU/mL.

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