CN103169075A - Fermented bean product fermented by lactobacillus plantarum ST-III and alpha-glucosidase inhibitor - Google Patents
Fermented bean product fermented by lactobacillus plantarum ST-III and alpha-glucosidase inhibitor Download PDFInfo
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Abstract
The invention discloses a use of lactobacillus plantarum ST-III in preparation of an alpha-glucosidase inhibitor or the preparation of a fermented bean product with alpha-glucosidase inhibition activity. The invention further discloses fermented soymilk or fermented beans with the alpha-glucosidase inhibition activity, which can be obtained by inoculating the lactobacillus plantarum ST-III into sterile soymilk or cooked soymilk and fermenting at the temperature of 25-40 DEG C. Water is added into the fermented beans for homogenizing, the pH value is regulated to 6.5-7.5, the boiling is performed for 10-60 minutes, cooling is performed, then solid-liquid separation is performed, and a liquid phase is taken so as to get a water-soluble extract of the fermented soymilk. The lactobacillus plantarum ST-III has high alpha-glucosidase inhibition activity and can be used in glucose-reducing foods, health care products or medicaments. As the oral glucose-reducing foods, the health care products or the medicaments, the high postprandial blood glucose of patients with diabetes can be reduced and the postprandial blood glucose fluctuation can be regulated.
Description
Technical field
The invention belongs to the fermented food field, be particularly related to Lactobacillus plantarum ST-III (Lb.plantarum CGMCC No.0847) to the alpha-glucosidase inhibition purposes of (comprising AMS, maltose and invertase), preparation has the inhibiting fermentation beans of alpha-glucosidase and/or method of soybean milk product and products thereof as fermented bacterium to use this bacterial strain, and the method and the product that prepare alpha-glucosidase restrainer from the fermentation beans of Lactobacillus plantarum ST-III fermentation preparation.
Background technology
In recent years, diabetes are widely current in the whole world, have become the chronic disease of the third-largest class serious harm human health after tumour, angiocardiopathy at present.Along with developing rapidly of China's economy, society, the change of diet structure and life style, pace of population aging's quickening, the illness rate of diabetes also presents the trend of quick increase.In the zooming while of diabetes prevalence, the awareness of China's diabetes, treatment rate and control rate are obviously on the low side.Diabetes have become the public health problem of a serious harm population of China health, and socio-economic development is produced to more and more serious impact.
According to the pathogenesis difference, diabetes are divided into type i diabetes (insulin-dependent) and type ii diabetes (non-insulin-depending type), and the latter accounts for more than 85% of diabetes sum.Up to now, the medicine for the treatment of type ii diabetes mainly is divided into according to the machine-processed difference for the treatment of: (1) Drugs Promoting Insulin Secretion: as sulfonylurea; (2) insulin sensitizer: as tetrahydrothiazole derivates such as guitar ancestors (troglitazon e) in the wrong; (3) alpha-glucosidase restrainer (α-GI): as acarbose etc.Due to α-GI have advantages of action temperature and lasting, toxic and side effect is little even nontoxic, therefore obtains more and more domestic and international researchers' favor.
Research shows, alpha-glucosidase restrainer (α-GI) can reduce diabetes patient's postprandial hyperglycemia effectively, a kind of as OHA, its place of having an effect is positioned at small intestine, the mode of action is the alpha-glucosidase activity suppressed in the small intestine body, thereby reaches the purpose that reduces postprandial hyperglycemia.If in user's diet, carbohydrate accounts for more than 50%, hypoglycemic effect is more obvious, so it is applicable to take crowd, especially middle and elderly diabetic patient that carbohydrate is staple food.Though the α-GI found at present can overcome some shortcomings of traditional antidiabetic drug, kind is relatively less, and people still constantly are devoted to the research of development of new α-GI, especially natural products.In recent years, from natural products, the α-GI of separation and extraction, mainly from herbaceous plant and actinomyces, only has minority about the hypoglycemic report of lactic acid bacteria product.
H.Fujita (J.Nutri.131:1211-1213,2001) reported that the flavouring fermented soya bean (Touchi) traditional from China adopt the mode of hot-water extraction, (rat cane sugar hydrolytic enzyme) the active extract that obtains thering is Inhibiting α-glucosidase, this extract has hypoglycemic activity in rat and hyperglycemia population; It is mixed with the alpha-amylase inhibitor phaseolamin that comes from navy bean (kidney bean) that U.S. Pat 7354606B2 discloses a kind of employing fermented soya bean extract, is used for the treatment of the method for hyperglycaemia.A.Momose (J.J.Soc.Food Tech.Sci, Feb, 2011) reported that the alpha-glucosidase restrainer be separated to from meso (fermented bean products used as flavoring that a kind of natural fermented method of employing of japanese traditional is produced) has the effect of regulating post-prandial glucose excursions.But no matter adopt which kind of method, its main active component all comes from the fermentation soybean that adopts natural method to prepare, in the method, the microbe species that participates in sweat is various, bacterium is arranged as Bacillus subtlis, mould is arranged as Aspergillus niger etc., form also unstable.In a lot of traditional zymotechniques, in order to prevent polluting, need to add the NaCl of high concentration, and the NaCl of high concentration has certain harmfulness for most cardiovascular patients, be not suitable as bread and cheese edible, mainly as flavoring, use; In traditional zymotic technique, because the microbe species that participates in sweat is various, the security of part microorganism and metabolite thereof remains further to be proved conclusively; The unstable rule that flora forms can't guarantee the stability of fermentation bean products quality prepared by different batches, more is unfavorable for manufacturing on a large scale.
Summary of the invention
Therefore, the technical problem to be solved in the present invention is exactly to suppress the active defect that traditional zymotic soybean product fermenting agent composition is unstable, fermentation time long, fermented bean products is not suitable as the bread and cheese use for having alpha-glucosidase, and a kind of alpha-glucosidase restrainer and the corresponding method of fermented bean products of leavening definite ingredients and therefrom preparation is provided.
After the inventor finds that Lactobacillus plantarum ST-III (CGMCC No.0847) grows in the soybean base-material, its product has strong alpha-glucosidase and suppresses active, thereby has completed the present invention.
Technical scheme one of the present invention is: the purposes of Lactobacillus plantarum ST-III in preparing alpha-glucosidase restrainer.
Technical scheme two of the present invention is: Lactobacillus plantarum ST-III has in preparation the purposes that alpha-glucosidase suppresses active fermented bean products.
Technical scheme three of the present invention is: have the method that alpha-glucosidase suppresses active fermented soybean milk a kind of the preparation, comprise the following steps: Lactobacillus plantarum ST-III is inoculated in aseptic soya-bean milk, 25-40 ℃ ferments 2 to 120 hours, obtains fermented soybean milk (or claiming fermented soybean milk).
Wherein, described soya-bean milk is to be prepared by the beans as the main food kind, as is selected from soybean, red bean, mung bean, broad bean, big white kidney bean and small white kidney bean, most preferably soybean.The preparation method of described soya-bean milk is conventional, as be prepared into soya-bean milk in soy bean milk making machine.Soya-bean milk obtains aseptic soya-bean milk after sterilizing, the sterilizing methods routine, as the thermophilic digestion sterilizing, 118 ℃ 15 minutes.The preferred solid content 3%-9% of described sterilizing soya-bean milk (w/w), most preferably soymilk concentration is 5-6% (w/w).The preferred 0.5%-5% of the inoculum concentration of Lactobacillus plantarum ST-III in aseptic soya-bean milk (v/v), most preferably 2% (v/v).Fermentation temperature is 25-40 ℃, preferred 35-37 ℃, and best is 37 ℃.Fermentation time 2 to 120 hours, preferably 24-72 hour, more preferably 24-30 hour.Lactobacillus plantarum ST-III is inoculated in aseptic soya-bean milk and ferments, and can be used as unique fermented bacterium, also can ferment with other mushrooms simultaneously, and preferred plant lactobacillus ST-III of the present invention is as unique fermented bacterium.
Technical scheme four of the present invention is: a kind of have alpha-glucosidase and suppress active fermented soybean milk, by the said method preparation and obtain.
Technical scheme five of the present invention is: have the method that alpha-glucosidase suppresses active fermentation beans a kind of the preparation, comprises the following steps: Lactobacillus plantarum ST-III is inoculated in the boiling beans, cultivates 13-15 days for 35-37 ℃, obtain the fermentation beans.
Wherein, described boiling beans is to be prepared by the beans as the main food kind, as is selected from soybean, red bean, mung bean, broad bean, big white kidney bean and small white kidney bean, most preferably soybean.The preparation method of described boiling beans is conventional, and the beans thermophilic digestion that bubble was sent out, reach the purpose of boiling sterilization, obtains the boiling beans.As 10-40 ℃ of soybean water soaked to 4-24 hour, then outwell Yu Shuihou, 110-121 ℃ obtain the boiling beans in boiling 15-40 minute.The preferred 0.5%-4% of the inoculum concentration of Lactobacillus plantarum ST-III in the boiling beans (v/w), most preferably 2-4% (v/v).Fermentation temperature is 25-40 ℃, preferred 35-37 ℃, and best is 37 ℃.Fermentation time is more than 1 day, preferably more than 10 days, and more preferably 13-15 days.Lactobacillus plantarum ST-III is inoculated in the boiling beans and ferments, and can be used as unique fermented bacterium, also can ferment with other mushrooms simultaneously, and preferred plant lactobacillus ST-III of the present invention is as unique fermented bacterium.
Technical scheme six of the present invention is: a kind of have alpha-glucosidase and suppress active fermentation beans, by the said method preparation and obtain.
Technical scheme seven of the present invention is: a kind of from described fermented soybean milk or the method for the alpha-glucosidase restrainer prepared from described fermentation beans, comprise the following steps: add water homogenate to obtain homogenate described fermentation beans, be adjusted to 6.5-7.5 by this homogenate or by the pH value of described fermented soybean milk, boil 10-60 minute, cooling rear Separation of Solid and Liquid is got liquid phase and get final product.
Wherein, the pH value is 6.5-7.5, preferably pH6.8-7.0.Boil 10-60 minute, preferably 30 minutes.Boil rear cool to room temperature and carry out Separation of Solid and Liquid.The method of Separation of Solid and Liquid is conventional method, as adopted multilayer gauze/nonwoven or squeeze and filter, or employing 3000-5000rpm (1,500g to 7,500g) centrifugal 15-20 minute, remove insoluble solid.The liquid phase of Separation of Solid and Liquid gained (water-soluble extract) is alpha-glucosidase restrainer, has the activity of Inhibiting α-glucosidase.Can adopt conventional method that this water-soluble extract is dried to powder, as adopted spray-drying (EAT 130-160 ℃, leaving air temp 110-130 ℃) or cryodesiccated mode, obtain concentrate dry powder.Be conventional method and the beans of wherein fermenting adds the method for water homogenate, as add the water of 5 to 10 times of quality to carry out homogenate.Homogenate can be carried out in refiner.
Technical scheme eight of the present invention is: a kind of alpha-glucosidase restrainer is obtained by described method preparation.
Technical scheme nine of the present invention is: above-mentioned fermentation beans of fermenting from Lactobacillus plantarum ST-III, prepare the purposes of alpha-glucosidase restrainer in antihypelipidemic foodstuff, health products or medicine.Preferably oral antihypelipidemic foodstuff, health products or medicine, reduce diabetes patient's postprandial hyperglycemia, regulates post-prandial glucose excursions.
In the present invention, Lactobacillus plantarum ST-III is prior art, is CGMCC No.0847.
The raw material that the present invention is used or reagent except special instruction, equal commercially available obtaining.
Than prior art, beneficial effect of the present invention is as follows:
1, the leavening that fermented soybean milk adopts forms clear and definite, can adopt single Lactobacillus plantarum ST-III, also can adopt the method for the mixed culture fermentations such as Lactobacillus plantarum ST-III and conventional yoghurt bacterium.
2, compare with the method for traditional zymotic bean product, adopt the present invention can obviously shorten fermentation time.
3, compare with the method for traditional zymotic bean product, adopt the fermentation beans of manufacture of the present invention to there is less salt, low sugar and high alpha-glucosidase inhibition activity, can be used as bread and cheese and directly eaten.
4, the fermented bean products that prepared by the present invention can be on a large scale, serialization production, and the product obtained can directly be used or as preparing on a large scale the raw material of alpha-glucosidase restrainer.
Thereby 5, adopt alpha-glucosidase restrainer prepared by the present invention to be directly used in to have and control carbohydrate and reach and control the blood glucose fluctuation purpose in the production of the product of intestinal absorption effect.
The accompanying drawing explanation
Below in conjunction with accompanying drawing, feature of the present invention and beneficial effect are described.
Fig. 1 shows that, under 37 ℃ of conditions, different strain is the inhibitory action of tunning to maltose in soybean milk.
Fig. 2 shows the inhibition activity of the tunning of Lactobacillus plantarum ST-III after 37 ℃ of fermentation variety classes soya-bean milk (middle growth) to maltose.
Fig. 3 shows under 37 ℃ of conditions, the impact of Lactobacillus plantarum ST-III different vaccination amount on soya-bean milk tunning inhibition maltase activity.
Fig. 4 shows tunning prepared at 37 ℃ of fermentation variable concentrations soya-bean milk by the Lactobacillus plantarum ST-III inhibitory action to maltose.
Fig. 5 shows that Lactobacillus plantarum ST-III different fermentations temperature suppresses the impact of maltase activity on the soya-bean milk tunning.
Fig. 6 shows under 37 ℃ of conditions, the impact of Lactobacillus plantarum ST-III different vaccination amount on fermented soybean product inhibition maltase activity.
Fig. 7 shows that Lactobacillus plantarum ST-III different fermentations temperature suppresses the impact of maltase activity on the fermented soybean product.
Fig. 8 shows the impact of Lactobacillus plantarum ST-III fermentation soybean concentrate on maltase activity.
Fig. 9 shows the impact of Lactobacillus plantarum ST-III fermented soybean milk concentrate on maltase activity.
The specific embodiment
The targeting enzymes of alpha-glucosidase restrainer comprises AMS, maltose and invertase etc.The present invention has done more detailed research to the inhibitory action of alpha-glucosidase in vitro to the Lactobacillus plantarum ST-III (Lactobacillus plantarum CGMCC No.0847) that is acknowledged as safety (GRAS), by setting up in-vitro screening model, inhibitory action to the tunning of lactic acid bacteria in bean product of separate sources to maltose compares, find that Lactobacillus plantarum ST-III has the inhibitory action strong to maltose, then further tested this bacterium and take the tunning of other beans after the base-material growth to the maltose inhibitory action.In the multiple beans as the main food kind through test, Lactobacillus plantarum ST-III is at the soybean milk of take through the soybean of boiling or sterilization after growth substrate, and the product of acquisition is the most obvious to the inhibition activity of maltose and invertase.
Being determined as follows the inhibiting rate of alpha-glucosidase in the present invention:
1. the mensuration of testing sample to the inhibiting rate of maltose
Testing sample to the inhibition activity of maltose as shown in following formula:
A wherein: blank sample is residual glucose content after the maltose reaction;
B: the original glucose content of blank sample;
C: fermented sample is residual glucose content after the maltose reaction;
D: be the corresponding content of the original glucose of soya-bean milk fermented sample.
The mensuration of the glucose content in above-mentioned formula all adopts glucose kit-oxidation enzyme process (GOD method).As shown in table 1, after according to form, order from left to right adds each reagent successively, 37 ℃ of water-bath 30Min, boiling water bath 5Min (cessation reaction) after taking out, after the centrifugal 2Min of 15,000rpm, reaction system A, B correspondence obtain testing sample A, B.By above-mentioned testing sample A, B, with after glucose assays kit (Puli's lema gene technology Co., Ltd) working solution mixes with the ratio of 1: 39 (v/v), 37 ℃ of water-bath 20Min, be determined at the absorbance at 550nm place.Wherein, the absorbance of reaction system A represents testing sample (blank or fermented sample) glucose content in system after the maltose reaction; The absorbance of reaction system B represents original glucose content in testing sample (blank or fermented sample).
Table 1. sample is to the inhibiting mensuration of maltose
2. the mensuration of testing sample to the inhibiting rate of invertase
Sample to the inhibitory action of invertase as follows shown in:
A wherein: blank sample is residual glucose content after the invertase reaction;
B: the original glucose content of blank sample;
C: fermented sample is residual glucose content after the invertase reaction;
D: be the original glucose content of soya-bean milk fermented sample.
The mensuration of the glucose content in above-mentioned formula all adopts glucose kit-oxidation enzyme process (GOD method).As shown in table 2, after according to form, order from top to bottom adds each reagent successively, 25 ℃ of water-bath 10Min, boiling water bath 5Min (cessation reaction) after taking out, after the centrifugal 2Min of 15,000rpm, reaction system A, B correspondence obtain testing sample A, B.By above-mentioned testing sample A, B, with after glucose assays kit (Puli's lema gene technology Co., Ltd) working solution mixes with the ratio of 1: 39 (v/v), 37 ℃ of water-bath 20Min, be determined at the absorbance at 550nm place.Wherein, wherein, the absorbance of reaction system A represents testing sample (blank or fermented sample) glucose content in system after the invertase reaction; The absorbance of reaction system B represents original glucose content in testing sample (blank or fermented sample).
Table 2. sample is to the inhibiting mensuration of invertase
| Numbering | Reaction system A | Reaction system B |
| 0.1M pH4.5 phosphate buffer | 550μL | 600μL |
| Invertase (40 μ g/mL) | 50μL | / |
| Testing sample (blank or fermented sample) | 100μL | 100μL |
| 0.1% (w/v) sucrose | 100μL | 100μL |
3. the mensuration of testing sample to the inhibiting rate of AMS (α-Amylase)
Sample to the inhibitory action of AMS as follows shown in:
A wherein: blank sample is residual glucose content after the AMS reaction;
B: the original glucose content of blank sample;
C: fermented sample is residual glucose content after the AMS reaction;
D: be the original glucose content of soya-bean milk fermented sample.
The mensuration of the glucose content in above-mentioned formula all adopts glucose kit-oxidation enzyme process (GOD method).As shown in table 3, after according to form, order from top to bottom adds each reagent successively, 37 ℃ of water-bath 10Min, boiling water bath 5Min (cessation reaction) after taking out, after the centrifugal 2Min of 15,000rpm, reaction system A, B correspondence obtain testing sample A, B.By above-mentioned testing sample A, B, with after glucose assays kit (Puli's lema gene technology Co., Ltd) working solution mixes with the ratio of 1: 39 (v/v), 37 ℃ of water-bath 20Min, be determined at the absorbance at 550nm place.Wherein, the absorbance of reaction system A represents testing sample (blank or fermented sample) glucose content in system after the maltose reaction; The absorbance of reaction system B represents original glucose content in testing sample (blank or fermented sample).
Table 3. sample is to the inhibiting mensuration of invertase
| Numbering | Reaction system A | Reaction system B |
| 0.1M pH4.5 phosphate buffer | 550μL | 600μL |
| α-Amylase(40μg/mL) | 50μL | |
| Testing sample (blank or fermented sample) | 100μL | 100μL |
| 0.1% (w/v) soluble starch (Sigma) | 100μL | 100μL |
Below with embodiment, further illustrate the present invention, but the present invention is not limited.The experimental technique of unreceipted actual conditions in the following example, usually according to normal condition, or the condition of advising according to manufacturer." room temperature " described in the present invention refers to the temperature of the operation room of being tested, and is generally 25 ℃.
The preparation of seed (fermented bacterium): respectively by L.plantarum ST-III, L.plantarumATCC14917, L.plantarum WCFS1, L.bulgaricus LB-340 (being provided by Danisco A/S BJ Rep Office), L.casei ATCC334, L.casei ATCC393 is inoculated in MRS liquid (Merck Co. Germany), S.thermophilus ST-BODY-3 (by section. the Chinese is gloomy to be provided) be inoculated in M17 (Merck Co. Germany), cultivate after 24 hours for 37 ℃, culture 9, centrifugal 10 minutes of 000rpm, supernatant discarded, thalline is with after sterile distilled water washing 2 times, with the sterile distilled water of former volume of culture, suspend, the seed (being working stock culture) of use obtains fermenting.
The preparation of soya-bean milk: after dry beans are weighed, the water that adds five times of quality is placed in 37 ℃ and soaks 8 hours, discards water, add by a certain percentage wet beans and water in soy bean milk making machine, make the soya-bean milk of required solid content, through 118 ℃ of 15Min sterilizings, obtain specifying the aseptic soya-bean milk of solid concentration.
As above method, adopt soybean to prepare the aseptic soya-bean milk that solid content is 5% (w/w).Then by the working stock culture of above-mentioned each bacterial strain of 2% (v/v) inoculum concentration access, 37 ℃ of cultivations, cultivate 24 hours, respectively at 16 hours and 24 hours sampling acquisition fermented soybean milks.
The pretreatment of fermented soybean milk testing sample: by after fermented soybean milk boiling water bath 5Min deactivation, the centrifugal 10Min of 15,000rpm, get supernatant, with 1M NaOH, is adjusted to pH6.80, and again 15, the centrifugal 10Min of 000rpm, get supernatant,, for test, is placed in-20 ℃ of preservations.
Test the inhibitory action to maltose through above-mentioned pretreated fermented soybean milk by described method.Wherein, the tunning after Lactobacillus plantarum ST-III fermentation soybean soya-bean milk is to maltose enzyme inhibition activity the highest (being shown in Fig. 1).Therefore, the present invention selects the working stock culture of Lactobacillus plantarum ST-III as the fermented bean products that is used for preparing alpha-glucosidase inhibitor, in following examples of the present invention, all take Lactobacillus plantarum ST-III as example.
Lactobacillus plantarum ST-III ferments the tunning of different beans soya-bean milk to the impact of maltose enzyme inhibition activity
The preparation of soya-bean milk: after dry beans are weighed, the water that adds five times of quality is placed in 37 ℃ and soaks 8 hours, discards water, add by a certain percentage wet beans and water in soy bean milk making machine, make the soya-bean milk of required solid content, through 118 ℃ of 15Min sterilizings, obtain specifying the aseptic soya-bean milk of solid concentration.
As above method, adopt respectively soybean, red bean, mung bean, broad bean, big white kidney bean and small white kidney bean to prepare the aseptic soya-bean milk that solid content is 5% (w/w).Then by 2% (v/v) inoculum concentration access Lactobacillus plantarum ST-III seed (being the working stock culture of embodiment 1 preparation), 37 ℃ of cultivations, cultivate 0 to 72 hour, obtains fermented soybean milk.
The pretreatment of fermented soybean milk testing sample: by after fermented soybean milk boiling water bath 5Min deactivation, the centrifugal 10Min of 15,000rpm, get supernatant, with 1M NaOH, is adjusted to pH6.80, and again 15, the centrifugal 10Min of 000rpm, get supernatant,, for test, is placed in-20 ℃ of preservations.
Test the inhibitory action to maltose through above-mentioned pretreated fermented soybean milk by described method.Wherein, the tunning after Lactobacillus plantarum ST-III fermentation soybean soya-bean milk is to maltose enzyme inhibition activity the highest (being shown in Fig. 2).Therefore, the present invention selects soybean as adopting Lactobacillus plantarum ST-III to prepare the more suitably raw material of alpha-glucosidase inhibitor, in following examples of the present invention, all take soybean as example.
Embodiment 3 fermented soybean milk condition optimizings
1) Lactobacillus plantarum ST-III different vaccination amount suppresses the impact of maltase activity on tunning
By Lactobacillus plantarum ST-III seed (working stock culture of embodiment 1 preparation), the inoculum concentration by 0.5%, 1%, 2%, 5% (v/v) accesses in the sterilizing soya-bean milk that solid content is 5% (w/w) respectively, and after inoculation, the viable count of Lactobacillus plantarum is 5 * 10
6to 5 * 10
7cfu/ml (every milliliter of CFU), 37 ℃ of cultivations, cultivate the fermented soybean milk of different time and measure its activity of inhibition to maltose, invertase and AMS by described method after pretreatment.
The fermented soybean milk obtained is 5-75% to the inhibiting rate of maltose, the results are shown in Figure 3.Inhibiting rate to invertase is 1-35%, and to AMS, without obvious inhibitory action, its inhibiting rate is 1-10%.Visible proper mode is that inoculum concentration is 2% (v/v).
2) Lactobacillus plantarum ST-III fermentation variable concentrations soya-bean milk impact on the maltose enzyme inhibition activity on tunning
In the sterilized soya-bean milk that the 2% inoculum concentration sterile working access solid content of respectively Lactobacillus plantarum ST-III seed (working stock culture) being take is 3%, 5%, 7%, 9%, 37 ℃ of cultivations, cultivate the fermented soybean milk of different time and measure its activity of inhibition to maltose, invertase and AMS by described method after pretreatment.
The fermented soybean milk obtained is 5-75% to the inhibiting rate of maltose, the results are shown in Figure 4.Inhibiting rate to invertase is 1-35%, and to AMS, without obvious inhibitory action, its inhibiting rate is 1-10%.Visible proper mode is that soymilk concentration is 5-6% (w/w).
3) Lactobacillus plantarum ST-III different fermentations temperature suppresses the impact of maltase activity on tunning
Lactobacillus plantarum ST-III seed (working stock culture) be take in the sterilized soya-bean milk that 2% inoculum concentration sterile working access solid content is 5%, 25-40 ℃ of cultivation, cultivate the fermented soybean milk of different time and measure its activity of inhibition to maltose, invertase and AMS by described method after pretreatment.
The fermented soybean milk obtained is 25-75% to the inhibiting rate of maltose, the results are shown in Figure 5.Inhibiting rate to invertase is respectively 1-35%, and to AMS, without obvious inhibitory action, its inhibiting rate is 1-10%.Visible proper mode is that fermentation temperature is 35-37 ℃, and best is 37 ℃; Fermentation time 24-30 hour.
The inhibitory action of fermentation soybean to maltose, invertase and AMS
The preparation of boiling soybean base-material: the proportional arrangement by soybean and water by 1: 5 (w/w), soak 8 hours in 37 ℃, discard water, 110-121 ℃ of boiling 30 minutes, standby after fully cooling.
1) Lactobacillus plantarum ST-III different vaccination amount suppresses the impact of maltase activity on tunning
In boiling soybean by Lactobacillus plantarum ST-III seed (working stock culture of embodiment 1 preparation) by the inoculum concentration access preparation of 0.5%, 1%, 2%, 4% (v/w), after inoculation, the viable count of Lactobacillus plantarum is 5 * 10
6to 2 * 10
7cfu/ml (every milliliter of CFU), 37 ℃ of cultivations, cultivate 1 to 15 day to obtain fermentation soybean.Cultivate the fermentation soybean of different time and measure after pretreatment its activity of inhibition to maltose, invertase and AMS.
The fermentation soybean pretreatment: fermentation soybean is added to deionization in the ratio of 1: 5 (w/w), after smashing homogenate, boil 20 minutes, to be cooled to room temperature, get supernatant and be adjusted to pH6.80 with 1M NaOH, the centrifugal 10Min of 15,000rpm, get supernatant,, for measuring, be placed in-20 ℃ of preservations.
Through above-mentioned pretreated fermentation soybean, measure respectively the inhibition activity to maltose, invertase and AMS.Prepared fermentation soybean is 25-90% to the inhibiting rate of maltose, the results are shown in Figure 6.Inhibiting rate to invertase is 5-35%, and to AMS, without obvious inhibitory action, its inhibiting rate is 1-10%.Visible proper mode is that inoculum concentration is 2-4% (v/v).
2) Lactobacillus plantarum ST-III different fermentations temperature suppresses the impact of maltase activity on tunning
In boiling soybean by Lactobacillus plantarum ST-III seed (working stock culture of embodiment 1 preparation) by the inoculum concentration access preparation of 0.5%, 1%, 2%, 4% (v/w), after inoculation, the viable count of Lactobacillus plantarum is 5 * 10
6to 2 * 10
7cfu/ml (every milliliter of CFU), 25-40 ℃ of cultivation, cultivate 1 to 15 day to obtain fermentation soybean.Cultivate the fermentation soybean of different time and measure after pretreatment its activity of inhibition to maltose, invertase and AMS.
Prepared fermentation soybean is 25-90% to the inhibiting rate of maltose, the results are shown in Figure 7.Inhibiting rate to invertase is 5-35%, and to AMS, without obvious inhibitory action, its inhibiting rate is 1-10%.Visible proper mode is that fermentation temperature is 35-37 ℃, fermentation time 13-15 days.
In embodiment 5 fermentation soybeans, high alpha-glucosidase suppresses the preparation of active concentrate
Inoculum concentration by Lactobacillus plantarum ST-III seed (working stock culture) by 4% (v/w) accesses in the boiling soybean prepared by the described method of embodiment 4, and after inoculation, the viable count of Lactobacillus plantarum is 2 * 10
7cfu/ml (every milliliter of CFU), fermentation temperature is 35 ℃, fermentation time 14 days.
The fermentation soybean of preparation adds the deionized water of 5 to 10 times of volumes in mass ratio, homogenate, stir on one side, add saturated Na on one side
2cO
3regulate the pH value and be 6.8 and boil 30 minutes, in the process of boiling, slowly stir.To be cooledly to after after room temperature, adopt the multilayer filtered through gauze, remove insoluble solid.The water-soluble extract obtained adopts spray-drying (130 ℃ of EATs, 130 ℃ of leaving air temps) mode to obtain concentrate dry powder.
After this concentrate dry powder adds deionized water by 1: 20 (w/w), fully dissolve, standing 30 minutes, get clear liquid and measure respectively its activity of inhibition to maltose, invertase and AMS by described method, the results are shown in Figure 8, inhibiting rate to maltose is 75%, inhibiting rate to invertase is 25%, and to AMS, without obvious inhibitory action, its inhibiting rate is 5%.
In embodiment 6 fermentation soybeans, high alpha-glucosidase suppresses the preparation of active concentrate
Inoculum concentration by Lactobacillus plantarum ST-III seed (working stock culture) by 4% (v/w) accesses in the boiling soybean prepared by the described method of embodiment 4, and after inoculation, the viable count of Lactobacillus plantarum is 2 * 10
7cfu/ml (every milliliter of CFU), fermentation temperature is 37 ℃, fermentation time 15 days.
The fermentation soybean of preparation adds the deionized water of 5 to 10 times of volumes in mass ratio, homogenate, stir on one side, adding 1mol/L NaOH to regulate the pH value on one side is 7.0.Boil 30 minutes, in the process of boiling, slowly stir.To be cooled to after after room temperature, centrifugal 15 minutes of 5000rpm, remove insoluble solid.The water-soluble extract obtained adopts spray-drying (160 ℃ of EATs, 110 ℃ of leaving air temps) to obtain concentrate dry powder.
After this concentrate dry powder adds deionized water by 1: 20 (w/w), fully dissolve, standing 30 minutes, get clear liquid and measure respectively its activity of inhibition to maltose, invertase and AMS by described method, inhibiting rate to maltose is 80%, inhibiting rate to invertase is 31%, to AMS, without obvious inhibitory action, its inhibiting rate is 7%.
In embodiment 7 fermentation soybeans, high alpha-glucosidase suppresses the preparation of active concentrate
Inoculum concentration by Lactobacillus plantarum ST-III seed (working stock culture) by 4% (v/w) accesses in the boiling soybean prepared by the described method of embodiment 4, and after inoculation, the viable count of Lactobacillus plantarum is 2 * 10
7cfu/ml (every milliliter of CFU), fermentation temperature is 36 ℃, fermentation time 15 days.
The fermentation soybean of preparation adds the deionized water of 5 to 10 times of volumes in mass ratio, homogenate, stir on one side, adding 1mol/LNaOH to regulate the pH value on one side is 6.5.Boil 30 minutes, in the process of boiling, slowly stir.To be cooled to after after room temperature, centrifugal 20 minutes of 3000rpm, remove insoluble solid.The water-soluble extract obtained adopts cryodesiccated mode to obtain concentrate dry powder.
After this concentrate dry powder adds deionized water by 1: 20 (w/w), fully dissolve, standing 30 minutes, get clear liquid and measure respectively its activity of inhibition to maltose, invertase and AMS by described method, inhibiting rate to maltose is 90%, inhibiting rate to invertase is 35%, to AMS, without obvious inhibitory action, its inhibiting rate is 10%.
In embodiment 8 fermented soybean milks, high alpha-glucosidase suppresses the preparation of active concentrate
In the sterilizing soya-bean milk that solid content is 5% (w/w), access 2% (v/v) Lactobacillus plantarum ST-III seed (working stock culture), 37 ℃ of fermentations are after 24 hours, and stir on one side, and adding 1mol/L NaOH to regulate the pH value on one side is 6.8.Boil 30 minutes, in the process of boiling, slowly stir.To be cooledly to after after room temperature, adopt squeeze and filter, remove insoluble solid.The water-soluble extract obtained adopts the mode of spray-drying (140 ℃ of EATs, 120 ℃ of leaving air temps) to obtain concentrate dry powder.
After this concentrate dry powder adds deionized water by 1: 20 (w/w), fully dissolve, standing 30 minutes, get clear liquid and measure respectively its activity of inhibition to maltose, invertase and AMS by described method, the results are shown in Figure 9, wherein, concentrate is 70% to the inhibiting rate of maltose, to the inhibiting rate of invertase, is 30%, to the inhibiting rate of AMS, is 5%.
In embodiment 9 fermented soybean milks, high alpha-glucosidase suppresses the preparation of active concentrate
In the sterilizing soya-bean milk that solid content is 5% (w/w), access 2% (v/v) Lactobacillus plantarum ST-III seed (working stock culture), 37 ℃ of fermentations are after 30 hours, and stir on one side, and adding 1mol/L NaOH to regulate the pH value on one side is 7.0.Boil 30 minutes, in the process of boiling, slowly stir.To be cooledly to after after room temperature, adopt multi-layer nonwoven fabrics to filter, remove insoluble solid.The water-soluble extract obtained adopts the mode of spray-drying (160 ℃ of EATs, 110 ℃ of leaving air temps) to obtain concentrate dry powder.
After this concentrate dry powder adds deionized water by 1: 20 (w/w), fully dissolve, standing 30 minutes, get clear liquid and measure respectively its activity of inhibition to maltose, invertase and AMS by described method, wherein, concentrate is 80% to the inhibiting rate of maltose, inhibiting rate to invertase is 35%, to the inhibiting rate of AMS, is 10%.
In embodiment 10 fermented soybean milks, high alpha-glucosidase suppresses the preparation of active concentrate
In the sterilizing soya-bean milk that solid content is 5% (w/w), access 2% (v/v) Lactobacillus plantarum ST-III seed (working stock culture), 37 ℃ of fermentations are after 26 hours, and stir on one side, and adding 1mol/L NaOH to regulate the pH value on one side is 7.5.Boil 30 minutes, in the process of boiling, slowly stir.To be cooled to after after room temperature, adopt 4000 centrifugal 18 minutes, remove insoluble solid.The water-soluble extract obtained adopts the mode of spray-drying (150 ℃ of EATs, 130 ℃ of leaving air temps) to obtain concentrate dry powder.
After this concentrate dry powder adds deionized water by 1: 20 (w/w), fully dissolve, standing 30 minutes, get clear liquid and measure respectively its activity of inhibition to maltose, invertase and AMS by described method, wherein, concentrate is 75% to the inhibiting rate of maltose, inhibiting rate to invertase is 25%, to the inhibiting rate of AMS, is 8%.
Embodiment 11 has the preparation that alpha-glucosidase suppresses active local flavor sugar-free fermented soybean milk
Get the 1Kg soybean, add 5Kg to contain 0.5% (w/v) Na
2cO
3deionized water, 37 ℃ are soaked 8 hours, the water that elimination is unnecessary, with after washed with de-ionized water 2 times, add ionized water to 16Kg, use soy bean milk making machine homogenate, adopt the mode of filtering to remove insoluble solid, obtain the soya-bean milk that soluble solid content is 6% (W/W).
After this soya-bean milk adds 0.15 (V/V) vanilla, homogeneous, 115 ℃ of sterilizations 15 minutes, to be cooled to below 40 ℃, after accessing the prepared Lactobacillus plantarum ST-III seed of 2% (V/V) embodiment 1,37 ℃ ferment 8 hours, i.e. acquisition has alpha-glucosidase and suppresses active local flavor sugar-free fermented soybean milk, and this fermented soybean milk, by described method, records its inhibitory action to maltose, invertase and AMS and is respectively 65%, 15%, 1%.
Get the 1Kg soybean, add 5Kg to contain 0.5% (w/v) Na
2cO
3deionized water, 35-37 ℃ is soaked 8 hours, the water that elimination is unnecessary, with after washed with de-ionized water 2 times, add ionized water to 16Kg, use soy bean milk making machine homogenate, adopt the mode of filtering to remove insoluble solid, obtain the soya-bean milk that soluble solid content is 6% (W/W).
After this soya-bean milk adds 0.16% (V/V) vanilla, homogeneous, 118 ℃ of sterilizations 15 minutes, to be cooled to below 40 ℃, after accessing the prepared Lactobacillus plantarum ST-III seed of 2% (V/V) embodiment 1,37 ℃ ferment 8 hours, i.e. acquisition has alpha-glucosidase and suppresses active local flavor sugar-free fermented soybean milk, and this fermented soybean milk, by described method, records its inhibitory action to maltose, invertase and AMS and is respectively 67%, 20%, 5%.
Embodiment 13 has the preparation that alpha-glucosidase suppresses active local flavor sugar-free fermented soybean milk
Get the 1Kg soybean, add 5Kg to contain 0.5% (w/v) Na
2cO
3deionized water, 35-37 ℃ is soaked 8 hours, the water that elimination is unnecessary, with after washed with de-ionized water 2 times, add ionized water to 16Kg, use soy bean milk making machine homogenate, adopt the mode of filtering to remove insoluble solid, obtain the soya-bean milk that soluble solid content is 6% (W/W).
After this soya-bean milk adds 0.18% (V/V) vanilla, homogeneous, 117 ℃ of sterilizations 15 minutes, to be cooled to below 40 ℃, after accessing the prepared Lactobacillus plantarum ST-III seed of 2% (V/V) embodiment 1,37 ℃ ferment 8 hours, i.e. acquisition has alpha-glucosidase and suppresses active local flavor sugar-free fermented soybean milk, and this fermented soybean milk, by described method, records its inhibitory action to maltose, invertase and AMS and is respectively 70%, 28%, 4%.
Press the method for embodiment 1, prepare the soya-bean milk that soluble solid content is 6% (W/W), this soya-bean milk add 1.6 (V/V) vanilla, and 1% (W/v) press the prepared alpha-glucosidase restrainer concentrate of embodiment 8 dry powder, after fully dissolving, homogeneous, 115 ℃ of sterilizations 15 minutes, to be cooled to below 40 ℃, sterile filling, can obtain and have alpha-glucosidase and suppress active local flavor sugar-free soybean milk, this fermented soybean milk, by described method, records its inhibitory action to maltose, invertase and AMS and is respectively 35%, 10%, 2%.
Embodiment 15 has the preparation that alpha-glucosidase suppresses active local flavor sugar-free soybean milk
Press the method for embodiment 1, prepare the soya-bean milk that soluble solid content is 6% (W/W), this soya-bean milk add 1.6% (V/V) vanilla, and 1% (W/v) press the prepared alpha-glucosidase restrainer concentrate of embodiment 8 dry powder, after fully dissolving, homogeneous, 116 ℃ of sterilizations 15 minutes, to be cooled to below 40 ℃, sterile filling, can obtain and have alpha-glucosidase and suppress active local flavor sugar-free soybean milk, this fermented soybean milk, by described method, records its inhibitory action to maltose, invertase and AMS and is respectively 40%, 15%, 5%.
Press the method for embodiment 1, prepare the soya-bean milk that soluble solid content is 6% (W/W), this soya-bean milk add 1.6% (V/V) vanilla, and 1% (W/v) press the prepared alpha-glucosidase restrainer concentrate of embodiment 8 dry powder, after fully dissolving, homogeneous, 118 ℃ of sterilizations 15 minutes, to be cooled to below 40 ℃, sterile filling, can obtain and have alpha-glucosidase and suppress active local flavor sugar-free soybean milk, this fermented soybean milk, by described method, records its inhibitory action to maltose, invertase and AMS and is respectively 37%, 12%, 4%.
Embodiment 17 has the preparation that alpha-glucosidase suppresses active local flavor sugar-free fermentation soybean
Get the 1Kg soybean, add 5Kg to contain 0.5% (w/v) Na
2cO
3deionized water, 36 ℃ are soaked 8 hours, the water that elimination is unnecessary, with after washed with de-ionized water 2 times, 120 ℃ of sterilizations 25 minutes, to be cooled to below 40 ℃, after accessing the prepared Lactobacillus plantarum seed ST-III of 2% (V/V) embodiment 1, 32 ℃ of fermentations are after 10 days, the vanillic aldehyde that adds 0.11% (v/w) salt taste essence and 0.16% (v/w), aseptic subpackaged, i.e. acquisition has alpha-glucosidase and suppresses active local flavor sugar-free fermentation soybean, this fermentation soybean is by described method, record it to maltose, the inhibitory action of invertase and AMS is respectively 75%, 25%, 8%.
Embodiment 18 has the preparation that alpha-glucosidase suppresses active local flavor sugar-free fermentation soybean
Get the 1Kg soybean, add 5Kg to contain 0.5% (w/v) Na
2cO
3deionized water, 35 ℃ are soaked 8 hours, the water that elimination is unnecessary, with after washed with de-ionized water 2 times, 118 ℃ of sterilizations 25 minutes, to be cooled to below 40 ℃, after accessing the prepared Lactobacillus plantarum seed ST-III of 2% (V/V) embodiment 1, 30 ℃ of fermentations are after 10 days, the vanillic aldehyde that adds 0.10% (v/w) salt taste essence and 0.15% (v/w), aseptic subpackaged, i.e. acquisition has alpha-glucosidase and suppresses active local flavor sugar-free fermentation soybean, this fermentation soybean is by described method, record it to maltose, the inhibitory action of invertase and AMS is respectively 70%, 20%, 5%.
Embodiment 19 has the preparation that alpha-glucosidase suppresses active local flavor sugar-free fermentation soybean
Get the 1Kg soybean, add 5Kg to contain 0.5% (w/v) Na
2cO
3deionized water, 37 ℃ are soaked 8 hours, the water that elimination is unnecessary, with after washed with de-ionized water 2 times, 121 ℃ of sterilizations 25 minutes, to be cooled to below 40 ℃, after accessing the prepared Lactobacillus plantarum seed ST-III of 2% (V/V) embodiment 1, 35 ℃ of fermentations are after 10 days, the vanillic aldehyde that adds 0.12% (v/w) salt taste essence and 0.18% (v/w), aseptic subpackaged, i.e. acquisition has alpha-glucosidase and suppresses active local flavor sugar-free fermentation soybean, this fermentation soybean is by described method, record it to maltose, the inhibitory action of invertase and AMS is respectively 75%, 25%, 8%.
Raw milk [by solid content 11.7% (w/w)] through centrifugal, purify after, adopt the 7KPa homogeneous, process 18 seconds for 68 ℃, be cooled to below 40 ℃, add successively maltodextrin, the prepared batchings such as alpha-glucosidase restrainer concentrate of embodiment 11 by table 4, stir, fully dissolve, after adopting the 18KPa homogeneous, process 18 seconds for 89 ℃, adopt spray-dired method, can produce 1,000kg and there is alpha-glucosidase and suppress active Milk powder for middle-aged and old people [moisture content≤3% (w/w)].This milk powder deionized water dissolving, be made into the solution of 10% (w/w), add 1mol/L HCl, after adjusting pH value is 4.7,4 ℃ standing 2 hours, centrifugal 20 minutes of 7500 * g, get clear liquid, after being 6.7 by 1mol/L NaOH adjusting pH value, recording its inhibitory action to maltose, invertase and AMS and be respectively 40%, 15%, 5%.
Table 4. has the formula that alpha-glucosidase suppresses active Milk powder for middle-aged and old people
Embodiment 21 has the preparation that alpha-glucosidase suppresses active Milk powder for middle-aged and old people
Raw milk [by solid content 11.7% (w/w)] through centrifugal, purify after, adopt the 5KPa homogeneous, process 15 seconds for 65 ℃, be cooled to below 40 ℃, add successively maltodextrin, the prepared batchings such as alpha-glucosidase restrainer concentrate of embodiment 11 by table 4, stir, fully dissolve, after adopting the 15KPa homogeneous, process 15 seconds for 88 ℃, adopt spray-dired method, can produce 1,000kg and there is alpha-glucosidase and suppress active Milk powder for middle-aged and old people [moisture content≤3% (w/w)].This milk powder deionized water dissolving, be made into the solution of 10% (w/w), add 1mol/L HCl, after adjusting pH value is 4.6,0 ℃ standing 2 hours, centrifugal 20 minutes of 7500 * g, get clear liquid, after being 6.6 by 1mol/L NaOH adjusting pH value, recording its inhibitory action to maltose, invertase and AMS and be respectively 35%, 10%, 1%.
Embodiment 22 has the preparation that alpha-glucosidase suppresses active Milk powder for middle-aged and old people
Raw milk [by solid content 11.7% (w/w)] through centrifugal, purify after, adopt the 10KPa homogeneous, process 20 seconds for 70 ℃, be cooled to below 40 ℃, add successively maltodextrin, the prepared batchings such as alpha-glucosidase restrainer concentrate of embodiment 11 by table 4, stir, fully dissolve, after adopting the 20KPa homogeneous, process 20 seconds for 90 ℃, adopt spray-dired method, can produce 1,000kg and there is alpha-glucosidase and suppress active Milk powder for middle-aged and old people [moisture content≤3% (w/w)].This milk powder deionized water dissolving, be made into the solution of 10% (w/w), add 1mol/L HCl, after adjusting pH value is 4.8,6 ℃ standing 2 hours, centrifugal 20 minutes of 7500 * g, get clear liquid, after being 6.8 by 1mol/L NaOH adjusting pH value, recording its inhibitory action to maltose, invertase and AMS and be respectively 40%, 15%, 5%.
Should be understood that, after having read foregoing of the present invention, those skilled in the art can make various changes or modifications the present invention, these equivalent form of values fall within the application's appended claims limited range equally.
Claims (10)
1. the purposes of Lactobacillus plantarum ST-III in preparing alpha-glucosidase restrainer.
2. Lactobacillus plantarum ST-III has in preparation the purposes that alpha-glucosidase suppresses active fermented bean products.
3. one kind prepares and has the method that alpha-glucosidase suppresses active fermented soybean milk, it is characterized in that, comprises the following steps: Lactobacillus plantarum ST-III is inoculated in aseptic soya-bean milk, and 25-40 ℃ of fermentation 2 to 120 hours, obtain fermented soybean milk.
4. method as claimed in claim 3, is characterized in that, described soya-bean milk is soybean milk.
5. one kind has alpha-glucosidase and suppresses active fermented soybean milk, it is characterized in that, by the described method preparation of claim 3 or 4 and obtain.
6. one kind prepares and has the method that alpha-glucosidase suppresses active fermentation beans, it is characterized in that, comprises the following steps: Lactobacillus plantarum ST-III is inoculated in the boiling beans, cultivates more than 1 day for 25-40 ℃, obtain the fermentation beans.
7. one kind has alpha-glucosidase and suppresses active fermentation beans, it is characterized in that, by method preparation claimed in claim 6 and obtain.
One kind from fermented soybean milk claimed in claim 5 or the method for the alpha-glucosidase restrainer prepared from fermentation claimed in claim 7 beans, it is characterized in that, comprise the following steps: add water homogenate to obtain homogenate fermentation beans claimed in claim 7, be adjusted to 6.5-7.5 by this homogenate or by the pH value of fermented soybean milk claimed in claim 5, boil 10-60 minute, cooling rear Separation of Solid and Liquid is got liquid phase and get final product.
9. an alpha-glucosidase restrainer, is characterized in that, by method preparation claimed in claim 8, obtained.
10. the purposes of alpha-glucosidase restrainer as claimed in claim 9 in antihypelipidemic foodstuff, health products or medicine.
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