CN113024676B - Lotus seed resistant starch with high bifidobacterium breve adhesion rate and preparation method and application thereof - Google Patents
Lotus seed resistant starch with high bifidobacterium breve adhesion rate and preparation method and application thereof Download PDFInfo
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Abstract
The invention relates to lotus seed resistant starch with high bifidobacterium breve adhesion rate and a preparation method and application thereof, wherein the preparation method of the lotus seed resistant starch comprises the following steps: extracting lotus seed starch, preparing starch milk, gelatinizing starch, and preparing and purifying resistant starch of crude lotus seeds. The lotus seed resistant starch has high adhesion rate of the bifidobacterium breve, can be applied to adhesion of the bifidobacterium breve, and further can be used for preparing a bifidobacterium breve preparation or a product so as to improve the effectiveness of the bifidobacterium breve-related product, enhance the resistance of the bifidobacterium breve to low pH value, prolong the viability of the bifidobacterium breve in food and improve the effect of the bifidobacterium breve-related food after entering a human body.
Description
Technical Field
The invention relates to lotus seed resistant starch with high bifidobacterium breve adhesion rate and a preparation method and application thereof.
Background
The lotus seed is a special food with rich nutritive value and homology of medicine and food, and is widely used for food processing and industrial production. The content of starch in the lotus seed starch accounts for more than 50% of the dry matter content, the lotus seed starch contains more than 42% of amylose, and the lotus seed starch has short main chain length, short branched chain and small branching degree, and is very easy to form lotus seed resistant starch after gelatinization and aging. The resistant starch is also called as resistant starch, is defined as the sum of starch which can not be absorbed and degraded in small intestine, and has the physiological functions of regulating intestinal metabolism, regulating blood sugar and blood fat, reducing the incidence rate of gallstones and the like. Resistant starches are currently divided into five categories: physical embedded starch (RS 1), enzymolysis-resistant starch granules (RS 2), retrogradation starch (RS 3), chemically modified starch (RS 4) and amylose-lipid complex (RS 5). Retrogradation starch (RS 3) is gelatinized starch, and starch aggregates are changed from disordered state into ordered and regular arrangement under the action of Van der Waals force and hydrogen bonding force. The lotus seed resistant starch LRS3 is prepared from lotus seeds, can promote the growth of probiotics, inhibit the propagation of harmful bacteria, and promote the generation of short-chain fatty acids and the absorption of mineral substances by regulating intestinal flora.
The bifidobacterium is one of important constituent members of human and animal intestinal flora, and has multiple important physiological functions of biological barrier, nutrition, anti-tumor, immunity enhancement, gastrointestinal function improvement, aging resistance and the like on human health as a physiological beneficial bacterium. Bifidobacterium breve is one of the species of Bifidobacterium, and it can enhance the ability of the human body to produce interferon itself, enhance the production of self-resistance, increase and repair cells damaged by bacterial invasion or medical action, and improve the ability and quality of life. Bifidobacterium breve has inhibitory effect on putrefaction and pathogenic bacteria, pathogenic Escherichia coli, and Staphylococcus aureus. In addition, bifidobacterium breve has effects of preventing constipation, enhancing immunity, and preventing intestinal tumor. Therefore, the bifidobacterium breve is an important beneficial microorganism in intestinal tracts, and can be prepared into a bifidobacterium breve product for people to use. At present, related products of the bifidobacteria comprise lactic acid products, capsules and the like, but the storage effectiveness of the live bacteria preparation is poor, and the live bacteria are ingested into a human body to be influenced by gastric acid, so that the effect of supplementing the bifidobacteria into the human body is reduced. The adhesion of the bacteria to the starch granules enhances the resistance of the bifidobacteria to low pH in vitro and prolongs the viability of these bacteria in the food product. Different bifidobacterium strains have different adhesion capacities to starch granules, and no reports are provided on the adhesion capacity of bifidobacterium breve to starch granules, and no reports are provided on resistant starch capable of improving the adhesion rate of bifidobacterium breve. In view of the above, there is a need for a lotus seed resistant starch with high adhesion rate of bifidobacterium breve, so as to improve the effectiveness of bifidobacterium breve-related products, enhance the resistance of bifidobacterium breve to low pH, prolong the viability of the bacteria in food, and improve the effect of bifidobacterium breve-related food after entering human body.
Disclosure of Invention
The invention aims to provide lotus seed resistant starch with high bifidobacterium breve adhesion rate and a preparation method and application thereof. Compared with other methods such as an autoclave method, a water bath method and the like, the lotus seed resistant starch prepared by the preparation method has higher bifidobacterium breve adhesion rate, is resistant starch with high bifidobacterium breve adhesion rate, can be used for adhesion of bifidobacterium breve, and is used for preparation of bifidobacterium breve preparations or products.
The purpose of the invention is realized by the following technical scheme:
a preparation method of lotus seed resistant starch with high bifidobacterium breve adhesion rate comprises the following process steps:
(1) Extracting lotus seed starch: adding water into fresh lotus, and crushing in a juice extractor, wherein the weight ratio of the fresh lotus to the water is 1; then, sieving the mixture by a 120-mesh sieve, standing the filtrate for 6 hours for precipitation, and then removing the supernatant; washing the lower layer precipitate with industrial alcohol for 3 times until the supernatant has no yellow oil, and discarding the supernatant; washing the lower layer precipitate with distilled water, standing for 4 hr, removing supernatant, oven drying at 45 deg.C for 4 hr to water content of 11.7% to obtain lotus seed starch;
(2) Preparing starch milk: putting the lotus seed starch prepared in the step (1) into a beaker, adding distilled water, and preparing starch milk with the concentration of 15%;
(3) Gelatinizing starch: placing the starch milk in a microwave oven, and heating for 2-4min under the condition that the power is 640W to gelatinize the starch;
(4) Preparing resistant starch of crude lotus seeds: taking out the gelatinized starch in the step (3), standing at room temperature for 30min, cooling to room temperature, and then putting the starch into a refrigerator at 4 ℃ for cooling for 24h to regenerate; taking out regenerated starch, drying at 65 ℃, crushing, and sieving by a 80-mesh sieve to obtain crude lotus seed resistant starch;
(5) And (3) purification: adding distilled water into the crude lotus seed resistant starch obtained in the step (4), adjusting the pH to 5.0-6.0 by using a disodium hydrogen phosphate-citric acid buffer solution with the pH of 6.0, adding excessive alpha-amylase (500U/g, the amount is converted according to the amount of starch), and hydrolyzing for 2 hours at 55 ℃; cooling to room temperature, adjusting pH to 3.0-4.0 with 4mol/L citric acid, adding excessive glucoamylase (5000U/L, calculated according to starch amount), hydrolyzing at 60 deg.C for 1 hr, and cooling to room temperature; heating hydrolyzed starch milk in 100 deg.C water bath for 5min, inactivating enzyme, centrifuging at 4000r/min for 10min, refrigerating at 4 deg.C for 20-30min, and removing supernatant; washing the precipitate with distilled water for three times at 4000r/min for each washing, centrifuging for 10min, and removing supernatant; washing the precipitate with 95%,85% and 75% ethanol, centrifuging at 4000r/min for 10min, and removing supernatant; and (3) obtaining lotus seed resistant starch precipitate, drying at 65 ℃, crushing, and sieving with a 80-mesh sieve to obtain the lotus seed resistant starch.
And (3) when the starch is gelatinized in the step (3), placing the starch milk in a microwave oven, and heating for 3min under the condition that the power is 640W.
The lotus seed resistant starch with high bifidobacterium breve adhesion rate prepared by the preparation method.
The lotus seed resistant starch is applied to adhesion of bifidobacterium breve. Further, the application in the preparation of a bifidobacterium breve preparation or product.
Compared with the prior art, the invention has the advantages that: the lotus seed resistant starch has high adhesion rate of the bifidobacterium breve, can be used for adhesion of the bifidobacterium breve, is used for preparing a bifidobacterium breve preparation or a product, and is used for improving the effectiveness of the bifidobacterium breve related product, enhancing the resistance of the bifidobacterium breve to low pH value, prolonging the viability of the bifidobacterium breve in food and simultaneously improving the effect of the bifidobacterium breve related food after entering human bodies.
Drawings
Figure 1 is the effect of LRS3 obtained by different preparation methods on b.
FIG. 2 is ESEM images of apparent structures of resistant starch of lotus seeds of MP-LRS3-1, MP-LRS3-2, WB-LRS3-1 and WB-LRS3-2, wherein, images A, B, C and D are ESEM images under 2000 times of microscopic conditions; graphs a, b, c, d are ESEM images at 5000 times microscopic condition.
FIG. 3 is an X-ray diffraction pattern of LRS3 prepared by the preparation method described in examples one to four.
Fig. 4 is an infrared spectrum of LRS3 prepared in examples one to four.
Detailed Description
The invention is described in detail below with reference to the drawings and examples:
the main experimental materials:
fresh lotus seeds, available from food products of greenfield (fujian) ltd, produced by fujian ning;
alpha-amylase, ANKOM technologies, USA;
glucoamylase, st louis Sigma ltd, usa;
the citric acid, the disodium hydrogen phosphate and the acetic acid are all domestic analytical purities.
Main apparatus and equipment:
MJ-60BM01A Mei mixer, guangdong Mei Living appliances manufacturing Co., ltd;
analytical balance, mettler-toledo instruments (shanghai) ltd;
m700 microwave oven, midea Inc.;
model PB-10 pH meter, saedolis scientific instruments (Beijing) Inc.;
SX-500 model autoclave, TOMY, japan;
EYELA FDU-1200 lyophilizer, kyoto Kai, inc., tokyo Rikai, japan;
uv spectrophotometer, yuniko (shanghai) instruments ltd;
an L-530 model desk-top high-capacity low-speed centrifuge, hunan Xiangyi laboratory Instrument development Co., ltd;
a THZ-82A7 water bath constant temperature oscillator, national china electrical limited;
101-0ES digital display electrothermal blowing dry box, jinan optical instruments and Equipment manufacturing Limited;
clean bench, suzhou clean equipment ltd;
a pulverizer, wuyihanna electric limited;
MalvernMastersizer2000 laser diffraction particle size analyzer, malvern mechanical equipment ltd, uk;
fourier infrared spectroscopy, nikoli corporation, usa (Nicolet);
Philips-XL30 Environment scanning Electron microscope, philips instruments, inc. of the Netherlands;
x' Pert Pro MPD X-ray diffractometer, philips instruments Inc. of the Netherlands.
The first embodiment is as follows:
a preparation method of lotus seed resistant starch MP-LRS3-1 with high bifidobacterium breve adhesion rate comprises the following process steps:
(1) Extracting lotus seed starch: adding water into fresh lotus, and crushing in a juice extractor, wherein the weight ratio of the fresh lotus to the water is 1; then, sieving the mixture by a 120-mesh sieve, standing the filtrate for 6 hours for precipitation, and then removing the supernatant; washing the lower layer precipitate with industrial alcohol for 3 times until the supernatant has no yellow oil, and discarding the supernatant; washing the lower layer precipitate with distilled water, standing for 4 hr, removing supernatant, oven drying at 45 deg.C for 4 hr to water content of 11.7%, to obtain lotus seed starch;
(2) Preparing starch milk: putting the lotus seed starch prepared in the step (1) into a beaker, adding distilled water, and preparing starch milk with the concentration of 15%;
(3) Microwave gelatinization of starch: placing the starch milk in a microwave oven, and heating for 3min under the condition that the power is 640W to gelatinize the starch;
(4) Preparing resistant starch of crude lotus seeds: taking out the gelatinized starch in the step (3), standing at room temperature for 30min, cooling to room temperature, and then putting the starch into a refrigerator at 4 ℃ for cooling for 24h to regenerate; taking out regenerated starch, drying in an electrothermal blowing dry box with the temperature of 65 ℃, crushing, and sieving with a 80-mesh sieve to obtain crude lotus seed resistant starch;
(5) And (3) purification: adding distilled water into the crude lotus seed resistant starch obtained in the step (4), adjusting the pH to 5.0-6.0 by using a disodium hydrogen phosphate-citric acid buffer solution with the pH of 6.0, adding excessive alpha-amylase, and hydrolyzing for 2 hours at 55 ℃; cooling to room temperature, adjusting pH to 3.0-4.0 with 4mol/L citric acid, adding excessive glucoamylase, hydrolyzing at 60 deg.C for 1 hr, and cooling to room temperature; heating hydrolyzed starch milk in 100 deg.C water bath for 5min, inactivating enzyme, centrifuging at 4000r/min for 10min, refrigerating at 4 deg.C for 20-30min, and removing supernatant; washing the precipitate with distilled water for three times at 4000r/min for each washing, centrifuging for 10min, and removing supernatant; washing the precipitate with 95%,85% and 75% ethanol, centrifuging at 4000r/min for 10min, and removing supernatant; and (3) obtaining lotus seed resistant starch precipitate, drying at 65 ℃, crushing, and sieving with a 80-mesh sieve to obtain the lotus seed resistant starch MP-LRS3-1.
In order to distinguish the lotus seed resistant starch obtained by different preparation methods, the lotus seed resistant starch obtained in the first embodiment is named as MP-LRS3-1, and the lotus seed resistant starch obtained in the second embodiment is named as MP-LRS3-2; the lotus seed resistant starch obtained in example three is named as WB-LRS3-1, the lotus seed resistant starch obtained in example four is named as WB-LRS3-2, and the lotus seed resistant starch obtained in example five is named as GP-LRS3.
Example two:
a preparation method of lotus seed resistant starch MP-LRS3-2 with high bifidobacterium breve adhesion rate comprises the following process steps:
(1) Extracting lotus seed starch: adding water into fresh lotus, and crushing in a juice extractor, wherein the weight ratio of the fresh lotus to the water is 1; then, sieving the mixture by a 120-mesh sieve, standing the filtrate for 6 hours for precipitation, and then removing the supernatant; washing the lower layer precipitate with industrial alcohol for 3 times until the supernatant has no yellow oil, and discarding the supernatant; washing the lower layer precipitate with distilled water, standing for 4 hr, removing supernatant, oven drying at 45 deg.C for 4 hr to water content of 11.7%, to obtain lotus seed starch;
(2) Preparing starch milk: putting the lotus seed starch prepared in the step (1) into a beaker, adding distilled water, and preparing starch milk with the concentration of 15%;
(3) Microwave gelatinization of starch: placing the starch milk in a microwave oven, and heating for 3min under the condition that the power is 640W to gelatinize the starch;
(4) Preparing resistant starch of crude lotus seeds: taking out the gelatinized starch in the step (3), standing at room temperature for 30min, cooling to room temperature, and then putting the starch into a refrigerator at 4 ℃ for cooling for 24h to regenerate; taking out regenerated starch, drying in a freeze dryer at-46 ℃ and 20Pa, crushing, and sieving with a 80-mesh sieve to obtain crude extraction lotus seed resistant starch;
(5) And (3) purification: adding distilled water into the crude lotus seed resistant starch obtained in the step (4), adjusting the pH to 5.0-6.0 by using a disodium hydrogen phosphate-citric acid buffer solution with the pH of 6.0, adding excessive alpha-amylase, and hydrolyzing for 2 hours at 55 ℃; then cooling to room temperature, adjusting pH to 3.0-4.0 with 4mol/L citric acid, adding excessive glucoamylase, hydrolyzing at 60 deg.C for 1 hr, and cooling to room temperature; heating hydrolyzed starch milk in 100 deg.C water bath for 5min, inactivating enzyme, centrifuging at 4000r/min for 10min, refrigerating at 4 deg.C for 20-30min, and removing supernatant; washing the precipitate with distilled water for three times at 4000r/min for each washing, centrifuging for 10min, and removing supernatant; washing the precipitate with 95%,85% and 75% ethanol, centrifuging at 4000r/min for 10min, and removing supernatant; and (3) obtaining lotus seed resistant starch precipitate, drying at 65 ℃, crushing, and sieving with a 80-mesh sieve to obtain the lotus seed resistant starch MP-LRS3-2.
The difference between the second embodiment and the first embodiment is mainly the drying operation of the retrograded starch. Example one employs drying in an electrically heated forced air drying oven set at 65 deg.C, while example two employs freeze drying in a freeze dryer at-46 deg.C, 20 Pa.
Example three:
a preparation method of lotus seed resistant starch WB-LRS3-1 comprises the following steps:
(1) Extracting lotus seed starch: adding water into fresh lotus, and crushing in a juice extractor, wherein the weight ratio of the fresh lotus to the water is 1; then, sieving the filtrate by a 120-mesh sieve, standing the filtrate for 6 hours for precipitation, and then removing the supernatant; washing the lower layer precipitate with industrial alcohol for 3 times until the supernatant has no yellow oil, and discarding the supernatant; washing the lower layer precipitate with distilled water, standing for 4 hr, removing supernatant, oven drying at 45 deg.C for 4 hr to water content of 11.7% to obtain lotus seed starch;
(2) Preparing starch milk: putting the lotus seed starch prepared in the step (1) into a beaker, adding distilled water, and preparing starch milk with the concentration of 30%;
(3) Gelatinizing starch in water bath: placing the starch milk in a constant temperature water bath, and heating at 80 deg.C for 20min to gelatinize starch.
(4) Preparing resistant starch of crude lotus seeds: taking out the gelatinized starch in the step (3), standing at room temperature for 30min, cooling to room temperature, and then putting the starch into a refrigerator at 4 ℃ for cooling for 24h to regenerate; taking out regenerated starch, drying in an electrothermal blowing dry box with the temperature of 65 ℃, crushing, and sieving with a 80-mesh sieve to obtain crude lotus seed resistant starch;
(5) And (3) purification: adding distilled water into the crude lotus seed resistant starch obtained in the step (4), adjusting the pH to 5.0-6.0 by using a disodium hydrogen phosphate-citric acid buffer solution with the pH of 6.0, adding excessive alpha-amylase, and hydrolyzing for 2 hours at 55 ℃; then cooling to room temperature, adjusting pH to 3.0-4.0 with 4mol/L citric acid, adding excessive glucoamylase, hydrolyzing at 60 deg.C for 1 hr, and cooling to room temperature; heating hydrolyzed starch milk in 100 deg.C water bath for 5min, inactivating enzyme, centrifuging at 4000r/min for 10min, refrigerating at 4 deg.C for 20-30min, and removing supernatant; washing the precipitate with distilled water for three times at 4000r/min for each washing, centrifuging for 10min, and removing supernatant; washing the precipitate with 95%,85% and 75% ethanol, centrifuging at 4000r/min for 10min, and removing supernatant; and (3) obtaining lotus seed resistant starch precipitate, drying at 65 ℃, crushing, and sieving with a 80-mesh sieve to obtain the lotus seed resistant starch WB-LRS3-1.
Example four:
a preparation method of lotus seed resistant starch WB-LRS3-2 comprises the following steps:
(1) Extracting lotus seed starch: adding water into fresh lotus, and crushing in a juice extractor, wherein the weight ratio of the fresh lotus to the water is 1; then, sieving the filtrate by a 120-mesh sieve, standing the filtrate for 6 hours for precipitation, and then removing the supernatant; washing the lower layer precipitate with industrial alcohol for 3 times until the supernatant has no yellow oil, and discarding the supernatant; washing the lower layer precipitate with distilled water, standing for 4 hr, discarding supernatant, oven drying at 45 deg.C for 4 hr to water content of 11.7% to obtain semen Nelumbinis starch;
(2) Preparing starch milk: putting the lotus seed starch prepared in the step (1) into a beaker, adding distilled water, and preparing starch milk with the concentration of 15%;
(3) Gelatinizing starch in water bath: placing the starch milk in a constant temperature water bath, and heating at 80 deg.C for 20min to gelatinize starch.
(4) Preparing resistant starch of crude lotus seeds: taking out the gelatinized starch in the step (3), standing at room temperature for 30min, cooling to room temperature, and then putting the starch into a refrigerator at 4 ℃ for cooling for 24h to regenerate; taking out regenerated starch, drying in an electrothermal blowing dry box with the temperature of 65 ℃, crushing, and sieving with a 80-mesh sieve to obtain crude lotus seed resistant starch;
(5) And (3) purification: adding distilled water into the crude lotus seed resistant starch obtained in the step (4), adjusting the pH to 5.0-6.0 by using a disodium hydrogen phosphate-citric acid buffer solution with the pH of 6.0, adding excessive alpha-amylase, and hydrolyzing for 2 hours at 55 ℃; then cooling to room temperature, adjusting pH to 3.0-4.0 with 4mol/L citric acid, adding excessive glucoamylase, hydrolyzing at 60 deg.C for 1 hr, and cooling to room temperature; heating hydrolyzed starch milk in 100 deg.C water bath for 5min, inactivating enzyme, centrifuging at 4000r/min for 10min, refrigerating at 4 deg.C for 20-30min, and removing supernatant; washing the precipitate with distilled water for three times at 4000r/min for each washing, centrifuging for 10min, and removing supernatant; washing the precipitate with 95%,85% and 75% ethanol, centrifuging at 4000r/min for 10min, and removing supernatant; and (3) obtaining lotus seed resistant starch precipitate, drying at 65 ℃, crushing, and sieving with a 80-mesh sieve to obtain the lotus seed resistant starch WB-LRS3-2.
The difference between the third and fourth examples is that the concentration of the starch milk prepared in step (2) is different, the concentration of the third starch milk in the example is 30%, and the concentration of the fourth starch milk in the example is 15%.
Example five:
a preparation method of lotus seed resistant starch GP-LRS3 with high bifidobacterium breve adhesion rate comprises the following process steps:
(1) Extracting lotus seed starch: adding water into fresh lotus, and crushing in a juice extractor, wherein the weight ratio of the fresh lotus to the water is 1; then, sieving the mixture by a 120-mesh sieve, standing the filtrate for 6 hours for precipitation, and then removing the supernatant; washing the lower layer precipitate with industrial alcohol for 3 times until the supernatant has no yellow oil, and discarding the supernatant; washing the lower layer precipitate with distilled water, standing for 4 hr, removing supernatant, oven drying at 45 deg.C for 4 hr to water content of 11.7% to obtain lotus seed starch;
(2) Preparing starch milk: putting the lotus seed starch prepared in the step (1) into a beaker, adding distilled water, and preparing starch milk with the concentration of 30%;
(3) Pressing and heat gelatinizing starch: placing the starch milk in an autoclave, and heating at 121 deg.C for 10min to gelatinize starch;
(4) Preparing resistant starch of crude lotus seeds: taking out the gelatinized starch in the step (3), standing at room temperature for 30min, cooling to room temperature, and then putting the starch into a refrigerator at 4 ℃ for cooling for 24h to regenerate; taking out the regenerated starch, drying in an electrothermal blowing dry box with the temperature of 65 ℃, crushing, and sieving by a 80-mesh sieve to obtain crude lotus seed resistant starch;
(5) And (3) purification: adding distilled water into the crude lotus seed resistant starch obtained in the step (4), adjusting the pH to 5.0-6.0 by using a disodium hydrogen phosphate-citric acid buffer solution with the pH of 6.0, adding excessive alpha-amylase, and hydrolyzing for 2 hours at 55 ℃; cooling to room temperature, adjusting pH to 3.0-4.0 with 4mol/L citric acid, adding excessive glucoamylase, hydrolyzing at 60 deg.C for 1 hr, and cooling to room temperature; heating hydrolyzed starch milk in 100 deg.C water bath for 5min, inactivating enzyme, centrifuging at 4000r/min for 10min, refrigerating at 4 deg.C for 20-30min, and removing supernatant; washing the precipitate with distilled water for three times at 4000r/min for each washing, centrifuging for 10min, and removing supernatant; washing the precipitate with 95%,85% and 75% ethanol, centrifuging at 4000r/min for 10min, and removing supernatant; and (3) obtaining lotus seed resistant starch precipitate, drying at 65 ℃, crushing, and sieving with a 80-mesh sieve to obtain the lotus seed resistant starch GP-LRS3.
Example six: adhesion test of Bifidobacterium breve
The adhesion ability of bifidobacterium breve to starch granules was determined by a coprecipitation method. Breve was first washed twice with 10mL of 0.1mol/L phosphate buffer (pH = 7), suspended in buffer at a concentration of about 10 7 cells mL -1 . Breve suspension (2 mL) and starch particle suspension (2mL, 10g/L) were thoroughly mixed in a test tube with a diameter of 1cm (0.1M PBS, pH 7.0), and the mixture was allowed to stand at room temperature for 1 hour to allow starch to precipitate. Then, 150. Mu.L of the sample was aspirated from 0.5cm below the liquid surface by a pipette at OD 540nm Optical density was measured. Phosphate buffer was used as a blank control, and 2 samples containing sterile starch were used as controls. Calculating the adhesion rate of the lotus seed resistant starch obtained in the first to fifth embodiments to bifidobacterium breve, wherein the formula for calculating the adhesion rate of the lotus seed resistant starch to bacteria is as follows:
in the adhesion rate formula, a is OD of lotus seed resistant starch with bifidobacterium breve 540nm (ii) a b is OD of lotus seed resistant starch without bifidobacterium breve 540nm (ii) a c is OD of lotus seed resistant starch with bifidobacterium breve 540nm 。
Highly adherent strains can utilize starch and thus adherence to starch in certain bacteria plays a very important role. Strains in which more than 70% of the cells adhered to the starch granules were considered to be highly adherent strains. Moderate adhesion rates ranged from 40-70%, while less than 40% were low adhesion. The adhesion rate of bifidobacterium breve of the lotus seed resistant starch obtained in the first to fifth examples is shown in table 1:
TABLE 1 Bifidobacterium adhesion rates of lotus seed resistant starch obtained in examples one to five
As shown in table 1, the LRS3 prepared in the second to fifth examples showed a moderate adhesion rate of 40-70% to b.breve, while the LRS3 prepared in the first example showed a high adhesion rate of 79.53% to b.breve. It can be seen that the adhesion rate of LRS3 prepared by microwave method is significantly higher than that of water bath method (p < 0.05) and autoclave method. Since the adhesion of the macromolecular starch polymer to bacteria is closely related to the steric hindrance of the macromolecular starch polymer, the change of the LRS3 spatial structure (such as the influence on the exposure degree of contact receptors, surface charge and the like) inevitably causes the difference of the adhesion effect, and the difference is probably also the reason that the LRS3 has better proliferation effect on the bifidobacterium breve than resistant starch from other sources. The adhesion of LRS3 to Bifidobacterium breve is most likely related to the synergistic interaction of the two.
Since LRS3 obtained in example five has the lowest adhesion rate to bifidobacterium breve compared with LRS3 obtained in examples one to four, no further study was conducted on the lotus seed resistant starch obtained in example five, and only other properties of LRS3 obtained in examples one to four were studied and the possible relationship between each property and the adhesion rate of bifidobacterium breve was analyzed.
Example seven: proliferation of Bifidobacterium breve
GLU basal medium with carbon source concentration of 20g/L, MP-LRS3-1, WB-LRS3-1 and HAMS test medium were prepared respectively. Aspirate 1mL of activated b.breve suspension into 9mL of sterile basal and test media at different concentrations. After anaerobic culture in a constant temperature incubator at 37 ℃ for 48h, the absorbance values of the fermentation mixtures were measured at intervals of 4h and 600nm, respectively, using a basal medium without added bacteria and a test medium as blanks, and 3 replicates were set for each treatment group. With the culture time as the abscissa, the bacterial suspension OD 600nm The values are plotted on the ordinate and a bifidobacteria growth curve is plotted.
The preparation method of the culture medium is conventional technology, and therefore, the preparation of the culture medium is not excessively described.
Different substrate binding modules located in bacteria may be involved in adhesion to insoluble substrates, which is one of the prerequisites for bifidobacteria to use RS as a carbon source. The relationship between the adhesion difference and interaction between RS and Bifidobacterium is not clear. The present invention uses MP-LRS3-1 and WB-LRS3-1 culture media as examples, and uses GLU culture medium and HAMS culture medium as comparison medium, to investigate whether the adhesion of Bifidobacterium breve to LRS3 and the proliferation of LRS3 promoting bacteria exist correlation.
The effect of LRS3 on b.breve proliferation process for different preparations is shown in fig. 1, where b.breve growth rate was substantially consistent in all media and slightly higher in GLU media during the 0-8h culture period. After 8h, B.breve in MP-LRS3-1 and WB-LRS3-1 media grew into logarithmic phase, and the growth rate was significantly higher than that in GLU and HAMS media, while the B.breve growth rate in GLU and HAMS media remained essentially unchanged. After 20h, the total number of the bifidobacterium breve in the culture media of the two control groups is close to that of the two groups of LRS3 culture media, the growth rates of the bifidobacterium breve in the four groups of culture media after 24h are all in a stable period, after 48h of culture, along with the occurrence of substrate exhaustion, the total number of the bacteria in the LRS3 group is close to that in the HAMS culture medium, but the total number of the bacteria in the GLU culture medium is slightly lower. The number of Bifidobacterium breve in HAMS medium was close to that in MP-LRS3-1 and WB-LRS3-1 media, but the number of Bifidobacterium breve in GLU medium was smaller.
The growth trend of b.breve in LRS3 medium was essentially consistent in both experiments, but the growth amount of MP-LRS3-1 was significantly higher than WB-LRS3-1 (p < 0.05). This is probably because the difference in the ability of b.breve to adhere to MP-LRS3-1 and WB-LRS3-1 is due to their structural properties, which may also be the reason why bifidobacterium breve strains have a higher availability of LRS3 substrate than resistant starch from other sources. Combining the adhesion and proliferation of b.breve and LRS3, MP-LRS3-1 may promote the proliferation of b.breve more significantly than WB-LRS3-1, which may be related to the surface structure and crystallinity shown by ESEM and X-ray diffraction results. This particular structure may provide attachment points to protect the growth and proliferation of b. In addition, the related literature reports that the relatively loose molecular arrangement of the amorphous region and the unstable spatial structure of the region are not favorable for the growth of bifidobacteria.
Example eight: ESEM scanning Electron microscopy
The dried LRS3 samples obtained in examples one to four were dispersed on a sample stage, and after gold plating, the morphology of each lotus seed resistant starch particle was observed using ESEM with a magnification of 2000 × and 5000 × respectively. The ESEM images of the apparent structures of MP-LRS3-1, MP-LRS3-2, WB-LRS3-1, WB-LRS3-2 are shown in FIG. 2.
Under the scanning observation of an environmental electron microscope, under the condition of 5000 times of microscopy, MP-LRS3-1, MP-LRS3-2, WB-LRS3-1 and WB-LRS3-2 all show irregular block structures, rough surfaces in a scale shape and a gully shape are shown, and a plurality of layering strips (arrows in figure 2) exist on the surfaces of the particles. Compared with WB-LRS3, MP-LRS3 surface contained less particle debris, probably due to the fact that amylose overflowed from the particles during gelatinization to generate a double helix structure after retrogradation by microwave, the crystalline regions of amylopectin disappeared and the amylose was recombined between particles.
Comparing (A) and (B) in FIG. 2, it can be seen that although MP-LRS3-2 has slightly more surface debris than MP-LRS3-1, the overall apparent structure of MP-LRS3-1 and MP-LRS3-2 is substantially the same, indicating that hot air drying and freeze drying have little effect on the structure of MP-LRS 3. Comparing (C) and (D) in FIG. 2, WB-LRS3-1 had a smoother surface than WB-LRS3-2, indicating that moisture content had an effect on the apparent structure of WB-LRS3. In addition, the surface structure of WB-LRS3 is relatively loose, more concave cavities appear, the formation of the structures is closely related to the preparation characteristics of the structures, and the physical and chemical properties of MP-LRS3 and WB-LRS3 and the adhesion of LRS3 and B.breve can be influenced. From the adhesion results, MP-LRS3 showed better adhesion ability than WB-LRS3.
The MP-LRS3 refers to lotus seed resistant starch prepared by a microwave method, namely MP-LRS3-1 or MP-LRS3-2, and the WB-LRS3 refers to lotus seed resistant starch prepared by a water bath method, namely WB-LRS3-1 or WB-LRS3-2.
Example nine: size of particle diameter
A certain amount of the dried LRS3 samples obtained in examples one to four were weighed into a sample cell of a laser particle size analyzer, 200mL of ultrapure water was added to distill the water dispersant, and the sample particles were destroyed by 2000Hz ultrasonic treatment for 3min to uniformly disperse the starch particles. When the light-shielding rate reached 15%, the particle size range and distribution were measured and three replicates were performed. The refractive indices of the dispersant and starch particles were 1.33 and 1.53, respectively.
The size and distribution of starch granules vary with the conditions of starch production, thus affecting the physicochemical properties and applications of the starch. The results show that the mean area diameter D (3,2) and mean volume diameter D (4,3) of the four groups of LRS3 samples are significantly increased (p < 0.05) compared to the native starch (see table 2). Furthermore, for the MP-LRS3-2 group, the maximum value of D (3, 2) reached 30.8 μm, whereas D (3, 2) for WB-LRS3-2 was only 19.4 μm.
In terms of D (0.9), MP-LRS3 was generally larger than WB-LRS3, indicating an increased number of starch macroparticles in MP-LRS 3. This may be due to the swelling and coagulation of the starch granules during the microwave power. However, the tendency of change of the specific surface area is just opposite to that of D (0.9), which indicates that MP-LRS3 has a larger particle size but a smaller specific surface area than WB-LRS3. In conjunction with previous adhesion results, we speculate that the degree of adhesion between LRS3 and b.breve is independent of specific surface area, but may be affected by particle size, particle surface morphology, or other more important factors.
Table 2 particle size of LRS3 obtained in examples one to four
a Results are expressed as mean ± standard deviation; values are the average of 3 replicates. The lower case letters in the same column indicate that the results differ significantly (p)<0.05)。
Example ten: wide angle X-ray diffraction
The LRS3 samples of the lotus seed resistant starch obtained in the first to fourth examples were measured by an X-ray diffractometer. The operating conditions of the X-ray diffractometer were: the tube pressure is 40kV, the current is 100mA, the emission slit is 0.25nm, the scanning angle is 2 theta =4-60 degrees, the scanning speed is 0.02 degrees/min, and the scanning mode is continuous scanning. The crystallinity of the samples was quantitatively calculated and analyzed with peakfittv 4.12:
C CL ＝S C /S T ×100(％)
SC CL ＝S SC /S T ×100(％)
C L ＝C CL +SC CL
wherein C is CL Is the proportion of the crystalline region, S c Is a crystalline region, S t Is the total area, SC CL Is the proportion of the sub-crystalline region, S sc Is a sub-crystalline region, C l Is the degree of crystallinity.
The crystal structure of starch granules mainly exhibits three types of patterns. One is a type a spectrum, with characteristic peaks at 2 θ =15 °, 17 °, 18 ° and 23 °, including most cereal starches, and the other is a type B spectrum, with distinct peaks at 2 θ =17 °, and weaker diffraction peaks at 2 θ =20 °, 22 ° and 24 °, including tuber starches; and the third is a C-type spectrum formed by mixing A type and B type, and is mainly present in leguminous plants. Starch is a mixture consisting mainly of type a and type B crystals and a small amount of type V crystals.
TABLE 3 comparison of the crystallinity of LRS3 obtained in examples one to four
4X-ray diffraction pattern of LRS3 of preparation method as can be seen from fig. 3, all LRS3 showed strong diffraction peaks at diffraction angle 2 θ =17 °, weak diffraction peaks at 2 θ =20 °, 22 ° and 24 °, and showed similar pattern trends, indicating that MP-LRS3 and WB-LRS3 are both starches of B-type crystal structure. The intensity of diffraction peaks at 18 ℃ and 19 ℃ for WB-LRS3 was reduced compared to MP-LRS 3. As can be seen from Table 3, the 4 kinds of lotus seed resistant starch have the highest crystallinity of MP-LRS3-1, and MP-LRS3-1 >. The contrast of the crystallinity shows that the microwave treatment hot air drying is more beneficial to the formation of a crystallization area than other 3 methods. The amorphous degree of MP-LRS3-2 and WB-LRS3-2 is higher, which is consistent with the infrared spectrum detection result. Previous studies also found that the heat treatment regime did not affect the crystal type of LRS3, but was closely related to the retrogradation regime. 4, the preparation methods all present similar maps, and the crystal forms are B types as can be seen from table 3, which shows that in the process of preparing the resistant starch, the molecular chains form a more stable crystal structure through chain breakage and recombination, and the crystallinity, the sub-crystallinity and the amorphous degree are basically unchanged.
In combination with the adhesion results of MP-LRS3-1 having the highest adhesion rate, it was concluded that it had high crystallinity and high adhesion. RS3 is reported to be formed from amylose with the appropriate chain length, i.e. too long or too short starch chains are not conducive to the formation of resistant starch crystals. In this context, MP-LRS3 is therefore subjected to a short gelatinization process (3 min) so that amylose having a suitable chain length is extruded from the starch granules and, after retrogradation, a double helix structure is formed. The mass and number of double helix structures generated after MP-LRS3-1 regeneration are significantly increased compared to the remaining 3, which is consistent with FI-IR results. Therefore, it is presumed that the double helix structure is more favorable for adhesion of bacteria.
Example eleven: fourier transform infrared spectroscopy (FT-IR)
The LRS3 samples obtained in the first to fourth examples and the potassium bromide powder were dried to a constant weight in an oven at 105 ℃ and then taken out. 2mg of lotus seed resistant starch sample is firstly added into an agate mortar, 150mg of potassium bromide powder is then added, and then the mixture is uniformly ground under an infrared lamp. Filling the ground powder into a tabletting mould, and pressing into a sheet shape. Scanning and measuring different resistant starch samples by using a Fourier infrared spectrometer of Nicolet, nykul, USA, setting parameters, and scanning wavelength ranges: 400-4000cm -1 And the scanning times are as follows: 16-32, resolution: 4cm -1 。
Infrared spectroscopy techniques allow the analysis of the molecular structure of starch granules, the variation of the intensity of the peaks of which reflects the characteristics of the conformational changes of the starch (including helical structure, chain conformation). The spectrum obtained by scanning 4 LRS3 with infrared spectrometer is shown in FIG. 4.
Table 4 hydrogen bond distribution of LRS3 obtained in examples one to four
As shown in the infrared spectrum of LRS3 prepared by different methods in FIG. 4, the vibrational absorption peaks of 4 LRS3 were substantially the same,this shows that different preparation methods have no difference in the influence on the type of LRS3 chemical groups, and different treatment methods (microwave method, water bath method) and different drying methods (hot air drying, freeze drying) do not cause the starch to generate new chemical groups and chemical bonds. The research shows that 3100-3700cm -1 The absorption peak in the spectral band is related to O-H stretching vibration, and 2926cm -1 ，1650cm -1 And 1410cm -1 The absorption peaks respectively represent C-H stretching vibration, COO-stretching vibration and C-H bending vibration, the vibration intensity of the radicals is increased, and the vibration absorption peak intensity corresponding to the infrared spectrogram is increased. As can be seen in fig. 4, at 3100 - 3700cm -1 Compared with MP-LRS3-1 and WB-LRS3-1, the absorption peak intensity of LRS3 after microwave treatment is higher than that of water bath treatment, which shows that water molecules enter starch granules to generate hydration and form stronger hydrogen bonds. It contributes to the stabilization of both chains in the double helix structure of type B starch, thereby inducing the formation of a more compact starch structure for bacterial adhesion. At 800-1200cm -1 The variation in the intensity of the absorption peaks within the range also shows a similar trend.
995cm wave band -1 、1022cm -1 、1047cm -1 Sensitive to resistant starch conformation, and is a characteristic band of infrared spectrum. At 995cm -1 、1047cm -1 Has an absorption peak related to the absorption of vibration of the hydrated crystal and the ordered structure at 1022cm -1 The absorption peak of (a) is related to the amorphous structure. Therefore, R can be used 1047/1022 (DO) indicates the degree of molecular order of LRS3, R 995/1022 (DD) characterizing the degree of double helix structure inside LRS3 molecules. As shown in table 4. The DO values of MP-LRS3-1, MP-LRS3-2, WB-LRS3-1, WB-LRS3-2 were 1.070,1.056,1.011,1.028, respectively, with the same tendency as the crystallinity of X-ray diffraction Table 3, indicating that the crystalline structure is built up by ordered starch chains. Wherein the DO value of MP-LRS3 is higher than that of WB-LRS3, indicating that MP-LRS3 contains more ordered starch chains. The ordered starch chains help to form crystalline domains, thereby creating a compact starch structure to which bacteria can adhere, consistent with the adhesion results described above. From various starchesThe DD value shows that the microwave treatment can promote the formation of a double helix structure more than the water bath treatment, and the water bath treatment has a weaker influence on the increase of double helix than the microwave treatment. The results show that all the peak types of the LRS3 prepared by the 4 methods show steamed bun peaks, and the degree of crystal order and the degree of double-helix structure are basically unchanged. Wherein the crystal of the MP-LRS3-1 has higher ordered degree and higher double-helix structure degree than other three LRS3.
In conclusion, the MP-LRS3-1 prepared by the invention is compared with MP-LRS3-2, WB-LRS3-1 and WB-LRS3-2
The MP-LRS3-1 is probably due to a rough strip-shaped surface structure, high crystallinity and a compact double-spiral structure, has the highest adhesion rate to B.breve, can be effectively used for adhering the bifidobacterium breve, and further, the MP-LRS3-1 can be applied to the preparation of bifidobacterium breve preparations or products, promotes the proliferation of the bifidobacterium breve, improves the effectiveness of bifidobacterium breve-related products, enhances the resistance of the bifidobacterium breve to low pH values, prolongs the viability of the bifidobacterium breve in foods and improves the effect of the bifidobacterium breve-related foods after entering human bodies.
It should be noted that, although the above embodiments have been described herein, the scope of the present invention is not limited thereby. Therefore, based on the innovative concepts of the present invention, the technical solutions of the present invention can be directly or indirectly applied to other related technical fields by making changes and modifications to the embodiments described herein, or by using equivalent structures or equivalent processes performed in the content of the present specification and the attached drawings, which are included in the scope of the present invention.
Claims (1)
1. The application of lotus seed resistant starch MP-LRS3-1 in preparing a bifidobacterium breve preparation is characterized in that: the lotus seed resistant starch MP-LRS3-1 has high bifidobacterium breve adhesion rate;
the preparation method of the lotus seed resistant starch MP-LRS3-1 comprises the following process steps:
(1) Extracting lotus seed starch: adding water into fresh lotus, and crushing in a juice extractor, wherein the weight ratio of the fresh lotus to the water is 1; then, sieving the mixture by a 120-mesh sieve, standing the filtrate for 6 hours for precipitation, and then removing the supernatant; washing the lower layer precipitate with industrial alcohol for 3 times until the supernatant has no yellow oil, and discarding the supernatant; washing the lower layer precipitate with distilled water, standing for 4 hr, discarding supernatant, oven drying at 45 deg.C for 4 hr to water content of 11.7% to obtain semen Nelumbinis starch;
(2) Preparing starch milk: putting the lotus seed starch prepared in the step (1) into a beaker, adding distilled water, and preparing starch milk with the concentration of 15%;
(3) Microwave gelatinization of starch: placing the starch milk in a microwave oven, and heating for 3min under the condition that the power is 640W to gelatinize the starch;
(4) Preparing resistant starch of crude lotus seeds: taking out the gelatinized starch in the step (3), standing at room temperature for 30min, cooling to room temperature, and then putting the starch into a refrigerator at 4 ℃ for cooling for 24h to regenerate; taking out regenerated starch, drying in an electrothermal blowing dry box with the temperature of 65 ℃, crushing, and sieving with a 80-mesh sieve to obtain crude lotus seed resistant starch;
(5) And (3) purification: adding distilled water into the crude lotus seed resistant starch obtained in the step (4), adjusting the pH to 5.0-6.0 by using a disodium hydrogen phosphate-citric acid buffer solution with the pH of 6.0, adding excessive alpha-amylase, and hydrolyzing for 2 hours at 55 ℃; then cooling to room temperature, adjusting pH to 3.0-4.0 with 4mol/L citric acid, adding excessive glucoamylase, hydrolyzing at 60 deg.C for 1 hr, and cooling to room temperature; heating hydrolyzed starch milk in 100 deg.C water bath for 5min, inactivating enzyme, centrifuging at 4000r/min for 10min, refrigerating at 4 deg.C for 20-30min, and removing supernatant; washing the precipitate with distilled water for three times at 4000r/min for each washing, centrifuging for 10min, and removing supernatant; washing the precipitate with 95%,85% and 75% ethanol, centrifuging at 4000r/min for 10min, and removing supernatant; and (3) obtaining lotus seed resistant starch precipitate, drying at 65 ℃, crushing, and sieving with a 80-mesh sieve to obtain the lotus seed resistant starch MP-LRS3-1.
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