CN1243101C - Process for preparing food function factor gamma-amino-butyric acid - Google Patents

Abstract

The present invention relates to a method for preparing a food function factor-gamma-amino-butanoic acid, particularly to a method which utilizes microorganism bioconversion to prepare gamma-amino-butanoic acid (GABA), and belongs to the biologic technical field of food. Lactic acid bacteria or a mixture of lactic acid bacteria and saccharomycete is used as a strain, L-sodium glutamate is used as a conversion substrate, a carbon source, a nitrogen source and inorganic salt are added to form a fermentation medium, and the gamma-amino-butanoic acid is prepared in a bioconversion mode by a fermentation method. When the lactic acid bacteria are cultured in MRS, PYG culture media at the temperature of 25 DEG C to 40 DEG C, the growth is good, and the lactic acid bacteria can be used as culture solution for seeds. During the fermentation preparation, the carbon source which is suitable for the culture media adopts dextrose, and the nitrogen source can adopt one of corn steep liquor, yeast cream, degreasing groundnut meal peanut meal, degreasing cottonseed cake powder, degreasing bean pulp powder, rice bran, casein, etc., or a mixture of some of the corn steep liquor, the yeast cream, the degreasing groundnut meal peanut meal, the degreasing cottonseed cake powder, the degreasing bean pulp powder, the rice bran, the casein, etc. After the fermentation, through detection, the concentration of the gamma-amino-butanoic acid in fermentation liquor can reach 300 to 500 mg/100 ml. A product obtained according to the present invention is safe and reliable, and has very high physiological functions.

Description

A preparation method of food functional factor gamma-aminobutyric acid

technical field

The invention relates to a method for preparing food functional factor γ-aminobutyric acid, in particular to a method for preparing γ-aminobutyric acid through microbial biotransformation, and belongs to the field of food biotechnology.

Background technique

γ-Aminobutyric acid (GABA) is a naturally occurring active amino acid, which is concentrated in the brain, spinal cord and liver of animals, and widely distributed in the plant kingdom. In animals, GABA, as an important neurotransmitter, has physiological activities such as lowering blood pressure, improving blood circulation in the brain, stabilizing the mind, strengthening the kidney and liver, inhibiting colorectal cancer, and improving lipid metabolism. Foods rich in GABA have good immune health effects and can be used as a functional food, so their research and development have also received attention. According to reports, GABA tea developed in Japan has good health care functions, and animal experiments have shown that it can safely and significantly reduce essential hypertension in mice. Clinical experiments also show that rice germ food rich in GABA has a prominent effect on improving menopause syndrome such as insomnia and anxiety.

In addition to the chemical synthesis method, the preparation method of GABA is mainly a biotransformation preparation method through the action of microbial enzymes on the substrate L-glutamic acid. It has been reported to produce GABA by tissue culture of Astragalus root, but the yield is low. Escherichia coli has high glutamate decarboxylase activity, and the quantitative determination of glutamate is based on the specific reaction of Escherichia coli decarboxylase. Therefore, there are many reports proposing to use E. coli cells to immobilize GABA to produce GABA, which can obtain a higher biotransformation rate. However, if E. coli is used to prepare GABA for food, there must be hidden dangers in safety and hygiene, and it cannot meet the high requirements of food hygiene. .

Recently, it has been reported that the acid-resistant growth mechanism of microorganisms such as lactic acid bacteria and Aspergillus is related to the activity of glutamic acid decarboxylase contained in them. For example, Lactobacillus brevis, Lactobacillus plantarum, and yeast may have glutamic acid decarboxylase activity. At the same time, lactic acid bacteria, yeast, and aspergillus are several important microorganisms widely used in the food industry, especially lactic acid bacteria are a type of microorganisms that are recognized as safe, and can easily meet the requirements of food grade, and the immune efficacy and nutrition of this type of bacteria The function has been extensively and in-depth researched, and has certain curative effects and functions. For example, lactic acid bacteria play an important role in regulating human microcirculation and improving immunity, and have a significant effect on inhibiting the proliferation of harmful bacteria, etc. Therefore, the use of lactic acid bacteria to develop GABA-rich foods has a good prospect, and there is no relevant research report in China.

Contents of the invention

The purpose of the present invention is to provide a method for preparing food functional factor γ-aminobutyric acid, and the specific content relates to a method for preparing γ-aminobutyric acid through biotransformation of lactic acid bacteria and yeast.

The preparation method of a kind of gamma-aminobutyric acid of the present invention is to use lactic acid bacteria (Lactobacillus plantarum, Streptococcus lactis, Lactococcus lactis, Lactobacillus casei) or lactic acid bacteria and yeast (Saccharomyces sp) to mix as strains, use L-sodium glutamate (L-MSG) is used as the conversion substrate, using appropriate carbon and nitrogen sources and inorganic salts to form a fermentation medium, and the fermentation method is used for biotransformation to prepare γ-aminobutyric acid.

Strains:

植物乳杆菌Lactobacillus plantarum AS1.550，植物乳杆菌Lactobacillus plantarum AS1.557，植物乳杆菌Lactocbacillusplantarum CNIFFI6003，植物乳杆菌Lactocbacillus plantarumCNIFFI6014，乳酸链球菌Streptococcus lactis CNIFFI6015，乳酸链球菌Streptococcus lactis CNIFFI6016，乳酸链球菌Streptococcuslactis CNIFFI6034，乳酸链球菌Streptococcus lactis CNIFFI6036，乳酸链球菌Streptococcus lactis CNIFFI6048，乳酸链球菌Streptococcus lactis CNIFFI6017，乳酸链球菌Streptococcus lactisCNIFFI6018，乳酸链球菌Streptococcus lactis CNIFFI6021，乳酸链球菌Streptococcus lactis CNIFFI6023，乳酸乳球菌乳脂亚种Lactococcus lactis subsp cremoris AS1.9, Lactobacillus casei subsp casei AS1.539.

Yeast: commercial Angel high-activity dry yeast (high sugar type), Hubei Angel Yeast Co., Ltd.

The above strains have been published, and the AS number indicates (China General Microbiological Culture Collection Center (CGMCC), Institute of Microbiology, Chinese Academy of Science; General Microbiology Center of China Microbiological Culture Collection Committee), and the subsequent number represents the preservation number. The CNIFFI number means (China National Institute of Food & Fermentation Industries; China Microbial Cultures Preservation Committee Industrial Microbial Cultures Preservation Management Center), and the subsequent number represents the preservation number.

For details, see Volume 27 of "Food and Fermentation Industry" published in March, 2002 (2001 Supplement): Catalog of Chinese Industrial Microorganisms, page IV, page 11, page 13.

Angel high-activity dry yeast is commercially available. For details, please refer to the product catalog of Hubei Yichang Angel Yeast Co., Ltd.

The above strains used in the present invention are preserved in the Food Science Research Office of the Food College of Jiangnan University, and the corresponding preservation numbers are SYFS1.001, SYFS1.002, SYFS1.003, SYFS1.004, SYFS1.005, SYFS1.006, SYFS1.007 , SYFS1.008, SYFS1.009, SYFS1.010, SYFS1.011, SYFS1.012, SYFS1.013, SYFS1.014, SYFS1.015. (Note: The SYFS number is the culture preservation number of the Food Science Research Office of the School of Food Science and Technology of Jiangnan University, and the sources of the bacteria are all the institutions mentioned above)

When a single lactic acid bacteria is used as the strain, SYFS1.008 and SYFS1.009 are better.

If lactic acid bacteria and yeast are mixed as strains, yeast and various lactic acid bacteria can be used in combination. Because yeast can hydrolyze protein to produce L-glutamic acid during fermentation and growth, the substrate L-sodium glutamate can be used less or not during fermentation. If the same amount of L-sodium glutamate substrate is still used, the conversion The effect will be better, and when mixed strains are used, the reaction mechanism is more complicated.

When using mixed strains, first culture the seeds separately, and then inoculate them into the fermentation medium at the same time and mix them for fermentation.

culture medium

Agar slant preservation medium (%): yeast extract 1.0%, glucose 1.5%, calcium carbonate 1.5%, agar 1.5%.

24# Lactic acid bacteria medium (%): 0.5% beef extract, 0.5% yeast extract, 1.0% glucose, 0.5% lactose, 1.0% peptone, 0.5% NaCl, 2.0% agar, pH6.8.

PY basal medium (%): contains peptone 0.5%, tryptone (Tryptone) 0.5%, yeast extract 1.0%, salt solution 4.0ml/100ml, (salt solution g/1000ml contains CaCl ₂ , 0.2; MgSO ₁ · 7H ₂ O, 0.48; K ₂ HPO ₄ , 1.0; KH ₂ PO ₄ , 1.0; NaHCO ₃ , 10.0; NaCl, 2.0), pH 6.8.

Fermented seed medium: MRS liquid medium.

GYP fermentation medium (%): glucose 1.0%, yeast extract 1.0%, peptone 0.5%, sodium acetate 0.2%, L-MSG 1.0%, MgSO ₄ 7H ₂ O 20ppm, MnSO ₄ 4H ₂ O 1ppm, NaCl 1ppm, FeSO ₄ ·7H ₂ O 1ppm, pH6.8.

Preparation Process

Strain storage:

The selected strains were passaged on agar slant preservation medium and 24# lactic acid bacteria medium according to the requirements, stored in the refrigerator at 4°C, transferred once every 2 to 4 weeks, and activated twice before use.

Seed culture:

It is carried out in GYP fermentation medium or MRS liquid medium, and is subcultured twice before fermentation, and cultivated for 16 hours.

Fermentation:

The 16-hour-cultivated seed culture solution was centrifuged to collect the bacteria, and the bacteria suspension (~10 ⁹ CFU/ml) was made with sterile water, and the inoculation amount was 0.2%~2.0%. Add different carbon sources and nitrogen sources In the PY medium, the filling volume in the 250ml Erlenmeyer flask is 50ml. The carbon sources are glucose, maltose, soluble starch, and their mixture with L-sodium glutamate (L-MSG), and the addition level is 0.5% to 3%. Nitrogen sources were peptone, tryptone, casein, casein phosphopeptide (CPP), defatted soybean meal, corn steep liquor, defatted cottonseed meal, defatted peanut meal, rice bran, fish meal, yeast extract, (NH ₄ ) ₂ SO _4. NaNO ₃ , the addition level is 0.5% to 5%. Static culture at 25°C to 40°C for 1 to 6 days to obtain a γ-aminobutyric acid solution.

In the obtained final fermentation liquid, the content of gamma-aminobutyric acid can reach 300-500 mg/100ml. After neutralization and deodorization, the solution can be directly dried to make powder, and the content of gamma-aminobutyric acid in the powder is more than 5%. It can also be further refined and purified to be used as a functional food additive.

Crystallization and purification

The gamma-aminobutyric acid solution is ion-exchanged with a strongly acidic styrene-based cation exchange resin, washed with ammonia water, extracted, purified, concentrated, crystallized, and dried to obtain the finished gamma-aminobutyric acid.

The method for experimental detection of GABA content is as follows:

Quantification of amino acids in fermentation broth: amino acid automatic analyzer method (Hitachi L-835 amino acid automatic analyzer), add 10ml 10% trichloroacetic acid (TCA) to 25ml fermentation broth, shake evenly, after appropriate dilution, filter through 0.45μm membrane , and the filtered supernatant was sent to an automatic amino acid analyzer for analysis.

Through experiment, obtained the main factor that influences gamma-aminobutyric acid (GABA) yield as follows:

Effect of different strains on GABA yield.

Add 1% L-MSG (more than 99.9% purity, Shanghai Guanshengyuan) to the GYP medium, and carry out fermentation experiments on some lactic acid bacteria, mixed samples of lactic acid bacteria and yeast, and test the fermentation broth after fermentation. The results showed that SYFS1.008 and SYFS1.009 had higher GABA yields. There are significant differences in the production of GABA by different strains.

Effect of fermentation time

Understand the fermentation process of lactic acid bacteria SYFS1.009, observe its growth curve in GYP medium, the inoculation amount is 0.5%, the results show that its fermentation speed is very fast, it enters the vigorous period in about 6 hours, and reaches the late logarithmic growth period after 14 hours. Therefore, the cultivation time of the culture medium as seeds is determined to be 14 to 16 hours to ensure vigorously growing thalli during the fermentation process, as shown in Figure 1. The experiment has achieved satisfactory results.

Effect of Inoculum Size

Observe the influence of the inoculum size of 0.2%～6.0% during fermentation, detect the fermented liquid after the culture liquid fermentation of different inoculum sizes, the result shows, inoculum size has no significant influence on the productive rate of GABA and the final acidity, and this may be because The base of the number of viable bacteria in the bacterial suspension was large, and the proliferation rate of lactic acid bacteria was very fast, which was not enough to cause a significant difference.

Effect of carbon source and concentration

The carbon source glucose (GLC), maltose (Malt), soluble starch (SS) and their mixture with L-MSG were added to the PY basal medium, and the addition amount was 1%, and the fermented liquid was detected after fermentation. As shown in Figure 2, the mixture of glucose and L-MSG has the best yield of GABA, and the cost of glucose is not high, so the mixture of glucose and L-MSG is selected as the carbon source, and the addition level is 0.5% to 3 %, the addition amount is respectively 0.5% and 1.0% as better.

Investigating the influence of glucose concentration, the results are shown in Figure 3. When the glucose concentration is low, the production of GABA is high. This is because lactic acid bacteria have a certain ability to produce acid, and the concentration of carbon source decreases, and the amount of acid production decreases. To a certain extent, it is related to the maintenance of high enzyme activity of the bacteria.

Effect of nitrogen source

The detection of the fermentation broth after the experiment showed that peptone (peptone), tryptone (tryptone), casein (casein), casein phosphopeptide (CPP), corn steep liquor, defatted cottonseed cake powder, defatted peanut cake powder, defatted soybean meal powder, Rice bran, fish meal, yeast extract, (NH ₄ ) ₂ SO ₄ , and NaNO ₃ can all be used as nitrogen sources, and the addition concentration is 0.5% to 5%, and a good GABA yield can be obtained (Fig. 4). Especially when organic nitrogen source is used as nitrogen source, the yield of GABA is higher, which is better than that of inorganic nitrogen source. In my country, the above-mentioned organic nitrogen source resources are very rich, among which soybean meal powder, casein, peanut cake powder, etc. are excellent edible protein resources. If they are used as raw materials to increase the content of active factor GABA through biotechnology, it will be It can prepare functional food with higher added value, and has a very broad prospect.

The advantage of the present invention is that it provides a method for preparing gamma-aminobutyric acid by biotransformation using lactic acid bacteria or a mixture of lactic acid bacteria and yeast, which shows that under the condition of 25°C to 40°C, the selected strains can be used in the MRS medium , PYG medium growth well. Through fermentation, the yield of GABA reaches 300-500mg/100ml. The suitable carbon source for the fermentation medium is glucose, and the nitrogen source is selected from one of corn steep liquor, yeast extract, defatted peanut cake powder, defatted cottonseed cake powder, defatted soybean meal powder, rice bran, casein, etc. or in combination. The selected strains have been recognized as meeting the requirements of food grade, ensuring food safety, and the lactic acid bacteria used in the experiment have been proved to have immune curative effect and nutritional function. physiological activity.

Description of drawings

Figure 1 The growth and fermentation process of SYFS1.009. Fig. 2 Effect of carbon source type on GABA accumulation. Figure 3 Effect of carbon source concentration on GABA production. Figure 4 Effect of some nitrogen sources on GABA accumulation.

Detailed ways

Example 1

Select lactic acid bacteria SYFS1.009 as the strain, carry out seed culture in GYP fermentation medium, the inoculum amount is 0.5%, and the culture time is 16 hours. The obtained seed culture solution is centrifuged to collect the bacteria, and the bacteria suspension seeds are made with sterile water solution, containing 10 ⁸ ～10 ⁹ CFU/ml (plate count). Put 50ml of fermentation medium in a 250ml Erlenmeyer flask, inoculate 0.2% suspended seed solution, add 1% glucose and 1% L-sodium glutamate as carbon source, add 1% casein as nitrogen source, and culture for 24 hours , to get gamma-aminobutyric acid solution, the GABA content is about 280mg/100ml.

Example 2

Lactic acid bacteria SYFS1.008 was selected, and the seed culture and fermentation conditions were the same as in Example 1 to obtain a γ-aminobutyric acid solution with a GABA content of about 200 mg/100 ml.

Example 3

Select lactic acid bacteria SYFS1.009 and Angel active dry yeast as strains, seed culture and fermentation conditions are the same as in Example 1, first carry out seed culture respectively, then inoculate 0.2% of the suspended seed liquid and mix as seed liquid for fermentation to obtain γ- Aminobutyric acid solution, GABA content is about 300 ~ 350mg/100ml. The spray-dried powder of the fermented liquid contains 4%-6% of gamma-aminobutyric acid.

Example 4

Lactic acid bacteria SYFS1.009 is selected as the strain, 0.5% L-sodium glutamate, 0.2% ordinary yogurt stabilizer, 3-6% sugar, etc. are added to 10% skimmed milk powder, and pasteurized. After the inoculation, the yoghurt containing 200mg/100ml of gamma-aminobutyric acid was obtained and the flavor was good.

Example 5

Lactic acid bacteria SYFS1.009 was selected as the strain, 5% rice germ and 0.5% defatted soybean meal powder were used to make a suspension, after homogenization treatment, 0.5% glucose and 0.5% L-MSG were added, sterilized, inoculated and fermented to obtain The product of γ-aminobutyric acid 150mg/100ml.

Claims (4)

1. the preparation method of a γ-An Jidingsuan is characterized in that with streptococcus acidi lactici (streptococcuslactis) CNIFFI6036 or CNIFFI6048; Or described streptococcus acidi lactici and yeast (Saccharomyces sp) Angel high activity dried yeast are used with as bacterial classification, with the L-Sodium Glutamate is conversion of substrate, add carbon source and nitrogenous source and inorganic salt and form fermention medium, the fermentation method bio-transformation prepares γ-An Jidingsuan

A. bacterial classification

Adopt described single streptococcus acidi lactici or streptococcus acidi lactici and yeast to use with as bacterial classification,

B. substratum

The agar slant storage medium is in the quality percentage: yeast extract paste 1.0%, and glucose 1.5%, lime carbonate 1.5%, agar 1.5%,

24# milk-acid bacteria substratum is in the quality percentage: extractum carnis 0.5%, and yeast extract paste 0.5%, glucose 1.0%, lactose 0.5%, peptone 1.0%, NaCl 0.5%, agar 2.0%, pH6.8,

The PY basic medium is in the quality percentage: inner protein peptone 0.5%, and tryptone 0.5%, yeast extract 1.0%, salts solution 4.0ml/100ml, described salts solution g/1000ml contains CaCl ₂, 0.2; MgSO ₄7H ₂O, 0.48; K ₂HPO ₄, 1.0; KH ₂PO ₄, 1.0; NaHCO ₃, 10.0; NaCl, 2.0, pH6.8,

The ferment-seeded substratum: the MRS liquid nutrient medium,

The GYP fermention medium is in the quality percentage: glucose 1.0%, yeast extract paste 1.0%, peptone 0.5%, sodium acetate 0.2%, L-Sodium Glutamate 1.0%, MgSO ₄7H ₂O 20ppm, MnSO ₄4H ₂O 1ppm, NaCl 1ppm, FeSO ₄7H ₂O 1ppm, pH6.8,

C. preparation technology

Bacterial classification is preserved: selected bacterial classification goes down to posterity on agar slant storage medium, 24# milk-acid bacteria substratum respectively as requested, 4 ℃ of preservations of refrigerator, and the switching of 2～4 weeks is once used front activating 2 times,

Seed culture: in GYP fermention medium or MRS liquid nutrient medium, carry out, go down to posterity through 2 times before the fermentation, cultivated 16 hours,

Fermentation: cultivate 16 hours the centrifugal collection thalline of seed culture fluid, make thallus suspension liquid 108CFU/ml～10 with sterilized water ⁹CFU/ml, inoculum size with 0.2%～2.0%, insert in the PY substratum that adds different carbon sources and nitrogenous source, liquid amount in the 250ml triangular flask is 50ml, carbon source is respectively glucose, maltose, Zulkovsky starch, or the mixture of they and L-Sodium Glutamate, interpolation level 0.5%～3%, nitrogenous source is respectively peptone, tryptone, casein, phosphopeptide caseinate, the defatted soybean meal powder, corn steep liquor, yeast extract paste, defatted peanut cake powder, absorbent cotton seed cake powder or rice bran, interpolation level 0.5%～5%, 25～40 ℃ leave standstill cultivation 1～6 day, obtain gamma-aminobutyric acid fermentation, in the resulting end of a period fermented liquid, alpha-aminobutyric acid content reaches 300mg/100ml～350mg/100ml, after this solution deodorization, convection drying is made pulvis, in the pulvis alpha-aminobutyric acid content more than 5%, or further refining purifying, as functional food additives

D. crystallization purifying

γ-An Jidingsuan solution is carried out ion-exchange with strongly acidic styrene type cation exchange resin, ammonia scrubbing, extraction, again through resin purification, concentrate, γ-An Jidingsuan gets product after the crystallization, drying.

2. the preparation method of a kind of γ-An Jidingsuan as claimed in claim 1 is characterized in that selecting for use when using bacterial classification with, and streptococcus acidi lactici and yeast carry out earlier seed culture respectively, and mixed fermentation in the fermention medium is gone in inoculation then.

3. the preparation method of a kind of γ-An Jidingsuan as claimed in claim 1 is characterized in that carbon source selects the mixture of glucose and L-Sodium Glutamate for use, and the addition of glucose and L-Sodium Glutamate is respectively 0.5% and 1.0%.

4. the preparation method of a kind of γ-An Jidingsuan as claimed in claim 1, it is characterized in that nitrogenous source selects for use in corn steep liquor, yeast extract paste, defatted peanut cake powder, absorbent cotton seed cake powder, defatted soybean meal powder, rice bran, the casein one or several to use with, concentration is 0.5%～5%.