阅读说明：本技术 一种生物发酵提取物减肥饼干及其制备方法 (Biological fermentation extract weight-losing biscuit and preparation method thereof ) 是由 霍美 于 2021-01-19 设计创作，主要内容包括：本发明公开了一种生物发酵提取物减肥饼干及其制备方法,包括酵母发酵黄花万寿菊提取物、花青素微胶囊、索马甜复合蛋白、面粉和水。利用酿酒酵母发酵黄花万寿菊,将发酵液浓缩,酶解破碎,获得酵母多肽、β-葡聚糖。β-葡聚糖具有良好的抗消化性,不易被人体消化,增强饱腹感；β-葡聚糖在肠道中与花青素在淸除自由基和提高总抗氧化能力方面具有协同效果,从而可提升花青素的生物利用率和生物有效性,并且β-葡聚糖也会提高花青素对油脂和胆酸盐的吸附,去脂排油,健康减肥；用长链脂肪酯化花青素,再结合脂质体,提升花青素的稳定性；酶解法制备微孔淀粉,再利用甲基纤维素对微孔淀粉进行修饰,制成花青素微胶囊保护花青素的活性。(The invention discloses a biological fermentation extract weight-losing biscuit and a preparation method thereof. Fermenting flos Tagetis Erectae with Saccharomyces cerevisiae, concentrating the fermentation liquid, performing enzymolysis and crushing to obtain yeast polypeptide and beta-dextran. The beta-glucan has good anti-digestion performance, is not easy to digest by human bodies, and enhances satiety; the beta-glucan has a synergistic effect with anthocyanin in intestinal tracts in the aspects of cleaning free radicals and improving the total oxidation resistance, so that the bioavailability and the bioavailability of the anthocyanin can be improved, and the beta-glucan can also improve the adsorption of the anthocyanin on grease and cholate, remove fat and discharge oil and reduce weight healthily; the long-chain fat is used for esterifying the anthocyanin, and the liposome is combined, so that the stability of the anthocyanin is improved; preparing microporous starch by an enzymolysis method, and modifying the microporous starch by using methylcellulose to prepare the anthocyanin microcapsules for protecting the activity of the anthocyanin.)

1. A biological fermentation extract weight reducing biscuit comprises 0.5-2 parts of yeast fermentation marigold extract, 0.5-2 parts of anthocyanin microcapsule, 0.1-1.5 parts of thaumatin composite protein, 20-30 parts of flour and 10-15 parts of water.

2. The biological fermentation extract weight-reducing biscuit according to claim 1, characterized in that: the thaumatin compound protein is a compound protein constructed by a genetic engineering means and is compounded with protein SUMO.

3. A preparation method of a biological fermentation extract weight-losing biscuit comprises the following specific steps:

the method comprises the following steps: fermenting flos Tagetis Erectae with Saccharomyces cerevisiae at 23-28 deg.C for 2-4 days;

step two: concentrating the fermentation liquid, performing enzymolysis in combination with ultrasonic crushing, and spray drying to obtain fermented extract of flos Tagetis Erectae;

step three: preparing a recombinant thaumatin compound protein gene by using a genetic engineering means, and extracting thaumatin compound protein by using escherichia coli fermentation;

step four: esterifying anthocyanin by an enzymatic esterification method, preparing a liposome compound, and treating corn starch by enzyme to prepare microporous starch; modifying the microporous starch by utilizing methylcellulose, and then wrapping the liposome composite anthocyanin by the microporous starch modified by the methylcellulose to prepare an anthocyanin microcapsule;

step five: mixing flos Tagetis Erectae extract, anthocyanin microcapsule, thaumatin composite protein, flour and water to prepare dough, adding yeast, fermenting at 20-28 deg.C for 4 hr, and baking in oven for 15min to obtain weight reducing biscuit.

4. The preparation method of the biological fermentation extract weight-reducing biscuit according to claim 3, characterized by comprising the following steps: the preparation method of the marigold fermented extract comprises the following steps: 15-20 parts of fermentation liquor: 1, concentrating, adjusting the pH to 6.5, preheating in a constant-temperature water bath at 60 ℃ for 30min, adding 3.5% (w/w) of yeast hydrolase for hydrolysis, hydrolyzing for 6h, and immediately heating in a boiling water bath for 15min to inactivate the enzyme; adding 2mol/L sodium hydroxide solution into the hydrolysate, adjusting pH to 8-9, extracting in water bath shaker at 50 deg.C for 2.0h, wherein the last 30min is placed in ultrasonic cleaning machine with ultrasonic power of 80% (200W).

5. The preparation method of the biological fermentation extract weight-reducing biscuit according to claim 3, characterized by comprising the following steps: preparing the esterified anthocyanin: mixing anthocyanidin and lauric acid uniformly, adding lipase for catalytic reaction after fully dissolving in ethanol solvent, reacting for 2-6 hours at 45 ℃, filtering enzyme by filter paper to stop the reaction after the reaction is finished, and removing the solvent by rotary evaporation to obtain the anthocyanin enzyme method esterification product.

6. The preparation method of the biological fermentation extract weight-reducing biscuit according to claim 5, characterized by comprising the following steps: preparation of the anthocyanin liposome complex: weighing a certain amount of lecithin, cholesterol and tween-80, adding into absolute ethyl alcohol, mixing uniformly and dissolving completely; evaporating at 50 deg.C under reduced pressure to remove ethanol to form film, and increasing vacuum degree for further pumping for 30 min; then adding a certain amount of anthocyanin solution dissolved in phosphate buffer solution (PBS; pH6.8, 0.05mol/L) to wash the membrane to obtain crude liposome solution; and (3) after ultrasonic treatment, sieving the crude liposome by adopting a 0.22-micron microporous filter membrane to obtain the anthocyanin liposome.

7. The preparation method of the biological fermentation extract weight-reducing biscuit according to claim 3, characterized by comprising the following steps: preparing the microporous starch: weighing dry corn native starch, placing in a container, adding sodium acetate buffer solution of a certain value, preheating in a water bath kettle at a certain temperature, and stirring with an electric stirrer; weighing complex enzyme (glucoamylase and alpha-amylase) according to the ratio of 4: preparing enzyme solution by using buffer solution in proportion of 1, transferring all the enzyme solution into starch suspension, timing, stirring and reacting for 4h, centrifuging the suspension for 5-10min at 2000 plus 5000rpm, washing with distilled water and centrifuging, repeating the steps for 2-5 times, drying the obtained starch in a vacuum drying box to constant weight, and crushing and sieving by using a crusher to obtain the microporous starch.

8. The preparation method of the biological fermentation extract weight-reducing biscuit according to claim 7, characterized by comprising the following steps: preparing the anthocyanin microcapsules: dissolving microporous starch in deionized water, stirring for 0.5h, dissolving methylcellulose (1/20-30 of microporous starch in mass) in a small amount of deionized water, performing ultrasonic treatment for 5min, adding into microporous starch solution, heating to 70 deg.C, and stirring for 2 hr; cooling to room temperature, adding the anthocyanin lipidosome, and continuously stirring for 3 hours to obtain the anthocyanin microcapsules.

9. The preparation method of the biological fermentation extract weight-reducing biscuit according to claim 3, characterized by comprising the following steps: the preparation of the recombinant thaumatin gene-containing escherichia coli comprises the following steps: obtaining an expression sequence of thaumatin through PCR amplification, linking a SUMO gene of fusion protein by using a Linker in front of a thaumatin promoter by using an overlap extension PCR method, respectively cutting a target protein expression sequence and pET9a plasmid by using Not I and EcoR I enzyme cutting sites, constructing escherichia coli expression plasmid by using T4 ligase, and transforming the plasmid into escherichia coli.

10. The preparation method of the biological fermentation extract weight-reducing biscuit according to claim 9, characterized by comprising the following steps: extracting the recombinant thaumatin: after the supernatant of the medium (100mL) was subjected to a needle treatment with 5mM sodium phosphate buffer (disodium phosphate 3.55g, sodium dihydrogen phosphate 3g in 5L water) at 4 ℃, the dialysate was passed through an SP-Sephadex C-25(Amersham biosciences) column (1.7 cm. times.5 cm) equilibrated with 5mM sodium phosphate buffer (pH7.0), recombinant thaumatin was eluted with a linear gradient of 0-500mM NaCl in 5mM sodium phosphate buffer (pH7.0), the protein fraction detected by SDS-PAGE was dialyzed against 5mM sodium phosphate buffer pH7.0, and the dialysate (12mL) was passed again through the SP-Sephadex C-25 column for further purification; bound proteins were eluted with a linear gradient of 20mM to 300mM NaCl in 5mM sodium phosphate buffer (pH 7.0); the recombinant thaumatin proteins were pooled and dialyzed against 5mM sodium phosphate buffer, pH 5.0.

Technical Field

The invention relates to the technical field of food, in particular to a biological fermentation extract weight-reducing biscuit and a preparation method thereof.

Background

The biscuit is a common snack, is convenient to eat and carry as a snack or added diet, and becomes an indispensable food in daily life. At present, most of biscuits in the market pay attention to taste, and more deeply pay attention to health, for example, the biscuits contain less fat and various nutrient substances necessary for human bodies, but the biscuits with special functions are rare, so the development potential of the biscuits is huge.

At present, the prior art also has the technology of weight-reducing biscuits, but most of the weight-reducing biscuits are simple combinations of weight-reducing materials, the weight-reducing effect is not ideal enough, and a series of side reactions are often accompanied when weight-reducing medicines are taken, so that the daily life of a patient is painful.

Therefore, it is necessary to design a biological fermentation extract weight-reducing biscuit with good health, anti-aging and weight-reducing effects and a preparation method thereof.

Disclosure of Invention

The invention aims to provide a biological fermentation extract weight-reducing biscuit and a preparation method thereof, which aim to solve the problems in the background technology.

In order to solve the technical problems, the invention provides the following technical scheme: a biological fermentation extract weight reducing biscuit comprises 0.5-2 parts of yeast fermentation marigold extract, 0.5-2 parts of anthocyanin microcapsule, 0.1-1.5 parts of thaumatin composite protein, 20-30 parts of flour and 10-15 parts of water.

According to the technical scheme, the thaumatin compound protein is a compound protein constructed by a genetic engineering means and compounded with protein SUMO.

A preparation method of a biological fermentation extract weight-losing biscuit comprises the following specific steps:

the method comprises the following steps: fermenting flos Tagetis Erectae with Saccharomyces cerevisiae at 23-28 deg.C for 2-4 days;

step two: concentrating the fermentation liquid, performing enzymolysis in combination with ultrasonic crushing, and spray drying to obtain fermented extract of flos Tagetis Erectae;

step three: preparing a recombinant thaumatin compound protein gene by using a genetic engineering means, and extracting thaumatin compound protein by using escherichia coli fermentation;

step four: esterifying anthocyanin by an enzymatic esterification method, preparing a liposome compound, and treating corn starch by enzyme to prepare microporous starch; modifying the microporous starch by utilizing methylcellulose, and then wrapping the liposome composite anthocyanin by the microporous starch modified by the methylcellulose to prepare an anthocyanin microcapsule;

step five: mixing flos Tagetis Erectae extract, anthocyanin microcapsule, thaumatin composite protein, flour and water to prepare dough, adding yeast, fermenting at 20-28 deg.C for 4 hr, and baking in oven for 15min to obtain weight reducing biscuit.

According to the technical scheme, the preparation of the marigold fermented extract comprises the following steps: 15-20 parts of fermentation liquor: 1, concentrating, adjusting the pH to 6.5, preheating in a constant-temperature water bath at 60 ℃ for 30min, adding 3.5% (w/w) of yeast hydrolase for hydrolysis, hydrolyzing for 6h, and immediately heating in a boiling water bath for 15min to inactivate the enzyme; adding 2mol/L sodium hydroxide solution into the hydrolysate, adjusting pH to 8-9, extracting in water bath shaker at 50 deg.C for 2.0h, wherein the last 30min is placed in ultrasonic cleaning machine with ultrasonic power of 80% (200W).

According to the technical scheme, the preparation of the esterified anthocyanin comprises the following steps: mixing anthocyanidin and lauric acid uniformly, adding lipase for catalytic reaction after fully dissolving in ethanol solvent, reacting for 2-6 hours at 45 ℃, filtering enzyme by filter paper to stop the reaction after the reaction is finished, and removing the solvent by rotary evaporation to obtain the anthocyanin enzyme method esterification product.

According to the technical scheme, the preparation of the anthocyanin lipidosome compound comprises the following steps: weighing a certain amount of lecithin, cholesterol and tween-80, adding into absolute ethyl alcohol, mixing uniformly and dissolving completely; evaporating at 50 deg.C under reduced pressure to remove ethanol to form film, and increasing vacuum degree for further pumping for 30 min; then adding a certain amount of anthocyanin solution dissolved in phosphate buffer solution (PBS; pH6.8, 0.05mol/L) to wash the membrane to obtain crude liposome solution; and (3) after ultrasonic treatment, sieving the crude liposome by adopting a 0.22-micron microporous filter membrane to obtain the anthocyanin liposome.

According to the technical scheme, the preparation of the microporous starch comprises the following steps: weighing dry corn native starch, placing in a container, adding sodium acetate buffer solution of a certain value, preheating in a water bath kettle at a certain temperature, and stirring with an electric stirrer; weighing complex enzyme (glucoamylase and alpha-amylase) according to the ratio of 4: preparing enzyme solution by using buffer solution in proportion of 1, transferring all the enzyme solution into starch suspension, timing, stirring and reacting for 4h, centrifuging the suspension for 5-10min at 2000 plus 5000rpm, washing with distilled water and centrifuging, repeating the steps for 2-5 times, drying the obtained starch in a vacuum drying box to constant weight, and crushing and sieving by using a crusher to obtain the microporous starch.

According to the technical scheme, the preparation of the anthocyanin microcapsules comprises the following steps: dissolving microporous starch in deionized water, stirring for 0.5h, dissolving methylcellulose (1/20-30 of microporous starch in mass) in a small amount of deionized water, performing ultrasonic treatment for 5min, adding into microporous starch solution, heating to 70 deg.C, and stirring for 2 hr; cooling to room temperature, adding the anthocyanin lipidosome, and continuously stirring for 3 hours to obtain the anthocyanin microcapsules.

According to the technical scheme, the preparation of the recombinant thaumatin gene-containing escherichia coli comprises the following steps: obtaining an expression sequence of thaumatin through PCR amplification, linking a SUMO gene of fusion protein by using a Linker in front of a thaumatin promoter by using an overlap extension PCR method, respectively cutting a target protein expression sequence and pET9a plasmid by using Not I and EcoR I enzyme cutting sites, constructing escherichia coli expression plasmid by using T4 ligase, and transforming the plasmid into escherichia coli.

According to the technical scheme, the extraction of the recombinant thaumatin protein comprises the following steps: after the supernatant of the medium (100mL) was subjected to a needle treatment with 5mM sodium phosphate buffer (disodium phosphate 3.55g, sodium dihydrogen phosphate 3g in 5L water) at 4 ℃, the dialysate was passed through an SP-Sephadex C-25(Amersham biosciences) column (1.7 cm. times.5 cm) equilibrated with 5mM sodium phosphate buffer (pH7.0), recombinant thaumatin was eluted with a linear gradient of 0-500mM NaCl in 5mM sodium phosphate buffer (pH7.0), the protein fraction detected by SDS-PAGE was dialyzed against 5mM sodium phosphate buffer pH7.0, and the dialysate (12mL) was passed again through the SP-Sephadex C-25 column for further purification; bound proteins were eluted with a linear gradient of 20mM to 300mM NaCl in 5mM sodium phosphate buffer (pH 7.0); the recombinant thaumatin proteins were pooled and dialyzed against 5mM sodium phosphate buffer, pH 5.0.

Compared with the prior art, the invention has the following beneficial effects: in the invention, the raw materials are mixed,

(1) edible flower yellow marigold contains various active substances such as nutrients, active protease, polyphenol compounds and the like, has the effects of dispelling wind, reducing pathogenic fire, reducing phlegm, relieving cough, calming liver and clearing heat, and is fermented by saccharomyces cerevisiae to degrade macromolecular substances so that the active ingredients are easier to absorb; the fermentation liquor is concentrated, enzymolysis and ultrasonic crushing are combined, and spray drying are carried out to obtain the yeast polypeptide and the beta-glucan, wherein the beta-glucan has good anti-digestion performance, is not easy to digest by human bodies, and can enhance satiety; the beta-glucan has a synergistic effect with anthocyanin in intestinal tracts in the aspects of cleaning DPPH free radicals and improving the total antioxidant capacity, so that the bioavailability and the bioavailability of the anthocyanin can be improved, and the metabolic transformation of the anthocyanin is promoted by improving intestinal flora and up-regulating II-phase metabolic enzyme expression; the beta-glucan can also improve the adsorption of the anthocyanin to grease and cholate, remove fat and discharge oil, and reduce weight healthily;

(2) the anthocyanin is esterified by using a long-chain fatty acid lauric acid enzyme method, and then the stability of the anthocyanin is improved by combining with the liposome; the corn starch is treated by the combination of the alpha-amylase and the glucoamylase, and the formed microporous starch has high water absorption rate and oil absorption rate; the microporous starch is modified by methylcellulose, so that particles can absorb fat-soluble substances more easily, then the liposome composite anthocyanin is wrapped by the microporous starch modified by the methylcellulose to prepare an anthocyanin microcapsule for protecting the activity of the anthocyanin, the anthocyanin microcapsule enables the anthocyanin to be relatively stable in gastric juice and can be matched with beta-glucan to play a role in satiety, and after the anthocyanin capsule enters an intestinal tract, the anthocyanin is slowly released in the intestinal juice to absorb grease and cholate in the intestinal tract, so that the effects of removing fat, discharging oil and losing weight healthily are achieved; the methyl cellulose can not be digested or absorbed in vivo, and can hold water by several times, so that satiety is caused; the methyl cellulose also has the function of relaxing bowels, and solves the problem of stubborn stool;

(3) thaumatin is a non-caloric sweetener, but loses sweetness over time at elevated temperatures. According to the method, the expression sequence of thaumatin disclosed on Genebank is 662bp, the expression sequence of thaumatin is obtained through PCR amplification, a Linker is used for linking the SUMO fusion protein in front of a thaumatin promoter by using an overlap extension PCR method so as to increase the stability and the expression quantity of the thaumatin, Not I enzyme cutting sites and EcoR I enzyme cutting sites are used for respectively cutting a target protein expression sequence and a pET9a plasmid, T4 ligase is used for constructing escherichia coli expression plasmid, the plasmid is transformed into escherichia coli, and a large amount of SUMO-thaumatin expressed by the escherichia coli is obtained through fermentation culture.

Detailed Description

The technical solutions in the embodiments of the present invention will be clearly and completely described below, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.

The invention provides the technical scheme that: a biological fermentation extract weight reducing biscuit comprises yeast fermentation flos Tagetis Erectae extract 0.5-2 parts, anthocyanidin microcapsule 0.5-2 parts, thaumatin composite protein 0.1-1.5 parts, flour 20-30 parts and water 10-15 parts.

According to the technical scheme, the thaumatin compound protein is a compound protein constructed by a genetic engineering means and compounded with protein SUMO.

A preparation method of a biological fermentation extract weight-losing biscuit comprises the following specific steps:

the method comprises the following steps: fermenting flos Tagetis Erectae with Saccharomyces cerevisiae at 23-28 deg.C for 2-4 days;

step two: concentrating the fermentation liquid, performing enzymolysis in combination with ultrasonic crushing, and spray drying to obtain fermented extract of flos Tagetis Erectae;

step three: preparing a recombinant thaumatin compound protein gene by using a genetic engineering means, and extracting thaumatin compound protein by using escherichia coli fermentation;

step four: esterifying anthocyanin by an enzymatic esterification method, preparing a liposome compound, and treating corn starch by enzyme to prepare microporous starch; modifying the microporous starch by utilizing methylcellulose, and then wrapping the liposome composite anthocyanin by the microporous starch modified by the methylcellulose to prepare an anthocyanin microcapsule;

step five: mixing flos Tagetis Erectae extract, anthocyanin microcapsule, thaumatin composite protein, flour and water to prepare dough, adding yeast, fermenting at 20-28 deg.C for 4 hr, and baking in oven for 15min to obtain weight reducing biscuit.

According to the technical scheme, the preparation of the marigold fermented extract comprises the following steps: 15-20 parts of fermentation liquor: 1, concentrating, adjusting the pH to 6.5, preheating in a constant-temperature water bath at 60 ℃ for 30min, adding 3.5% (w/w) of yeast hydrolase for hydrolysis, hydrolyzing for 6h, and immediately heating in a boiling water bath for 15min to inactivate the enzyme; adding 2mol/L sodium hydroxide solution into the hydrolysate, adjusting pH to 8-9, extracting in water bath shaker at 50 deg.C for 2.0h, wherein the last 30min is placed in ultrasonic cleaning machine with ultrasonic power of 80% (200W).

According to the technical scheme, the preparation of the esterified anthocyanin comprises the following steps: mixing anthocyanidin and lauric acid uniformly, adding lipase for catalytic reaction after fully dissolving in ethanol solvent, reacting for 2-6 hours at 45 ℃, filtering enzyme by filter paper to stop the reaction after the reaction is finished, and removing the solvent by rotary evaporation to obtain the anthocyanin enzyme method esterification product.

According to the technical scheme, the preparation of the anthocyanin lipidosome compound comprises the following steps: weighing a certain amount of lecithin, cholesterol and tween-80, adding into absolute ethyl alcohol, mixing uniformly and dissolving completely; evaporating at 50 deg.C under reduced pressure to remove ethanol to form film, and increasing vacuum degree for further pumping for 30 min; then adding a certain amount of anthocyanin solution dissolved in phosphate buffer solution (PBS; pH6.8, 0.05mol/L) to wash the membrane to obtain crude liposome solution; and (3) after ultrasonic treatment, sieving the crude liposome by adopting a 0.22-micron microporous filter membrane to obtain the anthocyanin liposome.

According to the technical scheme, the preparation of the microporous starch comprises the following steps: weighing dry corn native starch, placing in a container, adding sodium acetate buffer solution of a certain value, preheating in a water bath kettle at a certain temperature, and stirring with an electric stirrer; weighing complex enzyme (glucoamylase and alpha-amylase) according to the ratio of 4: preparing enzyme solution by using buffer solution in proportion of 1, transferring all the enzyme solution into starch suspension, timing, stirring and reacting for 4h, centrifuging the suspension for 5-10min at 2000 plus 5000rpm, washing with distilled water and centrifuging, repeating the steps for 2-5 times, drying the obtained starch in a vacuum drying box to constant weight, and crushing and sieving by using a crusher to obtain the microporous starch.

According to the technical scheme, the preparation of the anthocyanin microcapsules comprises the following steps: dissolving microporous starch in deionized water, stirring for 0.5h, dissolving methylcellulose (1/20-30 of microporous starch in mass) in a small amount of deionized water, performing ultrasonic treatment for 5min, adding into microporous starch solution, heating to 70 deg.C, and stirring for 2 hr; cooling to room temperature, adding the anthocyanin lipidosome, and continuously stirring for 3 hours to obtain the anthocyanin microcapsules.

According to the technical scheme, the preparation of the recombinant thaumatin gene-containing escherichia coli comprises the following steps: obtaining an expression sequence of thaumatin through PCR amplification, linking a SUMO gene of fusion protein by using a Linker in front of a thaumatin promoter by using an overlap extension PCR method, respectively cutting a target protein expression sequence and pET9a plasmid by using Not I and EcoR I enzyme cutting sites, constructing escherichia coli expression plasmid by using T4 ligase, and transforming the plasmid into escherichia coli.

According to the technical scheme, the extraction of the recombinant thaumatin protein comprises the following steps: after the supernatant of the medium (100mL) was subjected to a needle treatment with 5mM sodium phosphate buffer (disodium phosphate 3.55g, sodium dihydrogen phosphate 3g in 5L water) at 4 ℃, the dialysate was passed through an SP-Sephadex C-25(Amersham biosciences) column (1.7 cm. times.5 cm) equilibrated with 5mM sodium phosphate buffer (pH7.0), recombinant thaumatin was eluted with a linear gradient of 0-500mM NaCl in 5mM sodium phosphate buffer (pH7.0), the protein fraction detected by SDS-PAGE was dialyzed against 5mM sodium phosphate buffer pH7.0, and the dialysate (12mL) was passed again through the SP-Sephadex C-25 column for further purification; bound proteins were eluted with a linear gradient of 20mM to 300mM NaCl in 5mM sodium phosphate buffer (pH 7.0); the recombinant thaumatin proteins were pooled and dialyzed against 5mM sodium phosphate buffer, pH 5.0.

Example 1

A biological fermentation extract weight-reducing biscuit comprises 2 parts of yeast fermentation marigold extract, 1.5 parts of anthocyanin microcapsules, 0.8 part of thaumatin composite protein, 30 parts of flour and 12 parts of water.

According to the technical scheme, the thaumatin compound protein is a compound protein constructed by a genetic engineering means and compounded with protein SUMO.

A preparation method of a biological fermentation extract weight-losing biscuit comprises the following specific steps:

the method comprises the following steps: fermenting flos Tagetis Erectae with Saccharomyces cerevisiae at 28 deg.C for 2 days;

step two: concentrating the fermentation liquid, performing enzymolysis in combination with ultrasonic crushing, and spray drying to obtain fermented extract of flos Tagetis Erectae;

step three: preparing a recombinant thaumatin compound protein gene by using a genetic engineering means, and extracting thaumatin compound protein by using escherichia coli fermentation;

step four: esterifying anthocyanin by an enzymatic esterification method, preparing a liposome compound, and treating corn starch by enzyme to prepare microporous starch; modifying the microporous starch by utilizing methylcellulose, and then wrapping the liposome composite anthocyanin by the microporous starch modified by the methylcellulose to prepare an anthocyanin microcapsule;

step five: mixing flos Tagetis Erectae extract, anthocyanin microcapsule, thaumatin composite protein, flour and water to prepare dough, adding yeast, fermenting at 25 deg.C for 4 hr, and baking in oven for 15min to obtain weight reducing biscuit.

According to the technical scheme, the preparation of the marigold fermented extract comprises the following steps: 15-20 parts of fermentation liquor: 1, concentrating, adjusting the pH to 6.5, preheating in a constant-temperature water bath at 60 ℃ for 30min, adding 3.5% (w/w) of yeast hydrolase for hydrolysis, hydrolyzing for 6h, and immediately heating in a boiling water bath for 15min to inactivate the enzyme; adding 2mol/L sodium hydroxide solution into the hydrolysate, adjusting pH to 9, extracting in a water bath shaker at 50 deg.C for 2.0h, wherein the last 30min is placed in an ultrasonic cleaning machine with ultrasonic power of 80% (200W).

According to the technical scheme, the preparation of the esterified anthocyanin comprises the following steps: mixing anthocyanidin and lauric acid uniformly, adding lipase for catalytic reaction after fully dissolving in ethanol solvent, reacting for 3 hours at 45 ℃, filtering enzyme by filter paper to stop the reaction after the reaction is finished, and removing the solvent by rotary evaporation to obtain the anthocyanin enzyme method esterification product.

According to the technical scheme, the preparation of the anthocyanin lipidosome compound comprises the following steps: weighing a certain amount of lecithin, cholesterol and tween-80, adding into absolute ethyl alcohol, mixing uniformly and dissolving completely; evaporating at 50 deg.C under reduced pressure to remove ethanol to form film, and increasing vacuum degree for further pumping for 30 min; then adding a certain amount of anthocyanin solution dissolved in phosphate buffer solution (PBS; pH6.8, 0.05mol/L) to wash the membrane to obtain crude liposome solution; and (3) after ultrasonic treatment, sieving the crude liposome by adopting a 0.22-micron microporous filter membrane to obtain the anthocyanin liposome.

According to the technical scheme, the preparation of the microporous starch comprises the following steps: weighing dry corn native starch, placing in a container, adding sodium acetate buffer solution of a certain value, preheating in a water bath kettle at a certain temperature, and stirring with an electric stirrer; weighing complex enzyme (glucoamylase and alpha-amylase) according to the ratio of 4: preparing enzyme solution by using buffer solution according to the proportion of 1, transferring all the enzyme solution into starch suspension, timing, stirring and reacting for 4 hours, centrifuging the suspension for 8 minutes at 3000rpm, washing with distilled water and centrifuging, repeating the steps for 4 times, drying the obtained starch in a vacuum drying oven to constant weight, crushing by using a crusher and sieving to obtain the microporous starch.

According to the technical scheme, the preparation of the anthocyanin microcapsules comprises the following steps: dissolving microporous starch in deionized water, stirring for 0.5 hr, dissolving methylcellulose (1/30 mass of microporous starch) in small amount of deionized water, ultrasonic treating for 5min, adding into microporous starch solution, heating to 70 deg.C, and stirring for 2 hr; cooling to room temperature, adding the anthocyanin lipidosome, and continuously stirring for 3 hours to obtain the anthocyanin microcapsules.

According to the technical scheme, the preparation of the recombinant thaumatin gene-containing escherichia coli comprises the following steps: obtaining an expression sequence of thaumatin through PCR amplification, linking a SUMO gene of fusion protein by using a Linker in front of a thaumatin promoter by using an overlap extension PCR method, respectively cutting a target protein expression sequence and pET9a plasmid by using Not I and EcoR I enzyme cutting sites, constructing escherichia coli expression plasmid by using T4 ligase, and transforming the plasmid into escherichia coli.

According to the technical scheme, the extraction of the recombinant thaumatin protein comprises the following steps: after the supernatant of the medium (100mL) was subjected to a needle treatment with 5mM sodium phosphate buffer (disodium phosphate 3.55g, sodium dihydrogen phosphate 3g in 5L water) at 4 ℃, the dialysate was passed through an SP-Sephadex C-25(Amersham biosciences) column (1.7 cm. times.5 cm) equilibrated with 5mM sodium phosphate buffer (pH7.0), recombinant thaumatin was eluted with a linear gradient of 0-500mM NaCl in 5mM sodium phosphate buffer (pH7.0), the protein fraction detected by SDS-PAGE was dialyzed against 5mM sodium phosphate buffer pH7.0, and the dialysate (12mL) was passed again through the SP-Sephadex C-25 column for further purification; bound proteins were eluted with a linear gradient of 20mM to 300mM NaCl in 5mM sodium phosphate buffer (pH 7.0); the recombinant thaumatin proteins were pooled and dialyzed against 5mM sodium phosphate buffer, pH 5.0.

Example 2

A biological fermentation extract weight-reducing biscuit comprises 1 part of yeast fermentation marigold extract, 1 part of anthocyanin microcapsule, 0.5 part of thaumatin composite protein, 20 parts of flour and 10 parts of water.

According to the technical scheme, the thaumatin compound protein is a compound protein constructed by a genetic engineering means and compounded with protein SUMO.

A preparation method of a biological fermentation extract weight-losing biscuit comprises the following specific steps:

the method comprises the following steps: fermenting flos Tagetis Erectae with Saccharomyces cerevisiae at 23 deg.C for 3 days;

step two: concentrating the fermentation liquid, performing enzymolysis in combination with ultrasonic crushing, and spray drying to obtain fermented extract of flos Tagetis Erectae;

step three: preparing a recombinant thaumatin compound protein gene by using a genetic engineering means, and extracting thaumatin compound protein by using escherichia coli fermentation;

step four: esterifying anthocyanin by an enzymatic esterification method, preparing a liposome compound, and treating corn starch by enzyme to prepare microporous starch; modifying the microporous starch by utilizing methylcellulose, and then wrapping the liposome composite anthocyanin by the microporous starch modified by the methylcellulose to prepare an anthocyanin microcapsule;

step five: mixing flos Tagetis Erectae extract, anthocyanin microcapsule, thaumatin composite protein, flour and water to prepare dough, adding yeast, fermenting at 28 deg.C for 4 hr, and baking in oven for 15min to obtain weight reducing biscuit.

According to the technical scheme, the preparation of the marigold fermented extract comprises the following steps: and (3) mixing the fermentation liquor 15: 1, concentrating, adjusting the pH to 6.5, preheating in a constant-temperature water bath at 60 ℃ for 30min, adding 3.5% (w/w) of yeast hydrolase for hydrolysis, hydrolyzing for 6h, and immediately heating in a boiling water bath for 15min to inactivate the enzyme; adding 2mol/L sodium hydroxide solution into the hydrolysate, adjusting pH to 8, extracting in a water bath shaker at 50 deg.C for 2.0h, wherein the last 30min is placed in an ultrasonic cleaning machine with ultrasonic power of 80% (200W).

According to the technical scheme, the preparation of the esterified anthocyanin comprises the following steps: mixing anthocyanidin and lauric acid uniformly, adding lipase for catalytic reaction after fully dissolving in ethanol solvent, reacting for 5 hours at 45 ℃, filtering enzyme by filter paper to stop the reaction after the reaction is finished, and removing the solvent by rotary evaporation to obtain the anthocyanin enzyme method esterification product.

According to the technical scheme, the preparation of the anthocyanin lipidosome compound comprises the following steps: weighing a certain amount of lecithin, cholesterol and tween-80, adding into absolute ethyl alcohol, mixing uniformly and dissolving completely; evaporating at 50 deg.C under reduced pressure to remove ethanol to form film, and increasing vacuum degree for further pumping for 30 min; then adding a certain amount of anthocyanin solution dissolved in phosphate buffer solution (PBS; pH6.8, 0.05mol/L) to wash the membrane to obtain crude liposome solution; and (3) after ultrasonic treatment, sieving the crude liposome by adopting a 0.22-micron microporous filter membrane to obtain the anthocyanin liposome.

According to the technical scheme, the preparation of the microporous starch comprises the following steps: weighing dry corn native starch, placing in a container, adding sodium acetate buffer solution of a certain value, preheating in a water bath kettle at a certain temperature, and stirring with an electric stirrer; weighing complex enzyme (glucoamylase and alpha-amylase) according to the ratio of 4: preparing enzyme solution by using buffer solution according to the proportion of 1, transferring all the enzyme solution into starch suspension, timing, stirring and reacting for 4h, centrifuging the suspension for 5min at 3000rpm, washing with distilled water and centrifuging, repeating the steps for 3 times, drying the obtained starch in a vacuum drying oven to constant weight, crushing by using a crusher and sieving to obtain the microporous starch.

According to the technical scheme, the preparation of the anthocyanin microcapsules comprises the following steps: dissolving microporous starch in deionized water, stirring for 0.5 hr, dissolving methylcellulose (1/20 mass of microporous starch) in small amount of deionized water, ultrasonic treating for 5min, adding into microporous starch solution, heating to 70 deg.C, and stirring for 2 hr; cooling to room temperature, adding the anthocyanin lipidosome, and continuously stirring for 3 hours to obtain the anthocyanin microcapsules.

According to the technical scheme, the preparation of the recombinant thaumatin gene-containing escherichia coli comprises the following steps: obtaining an expression sequence of thaumatin through PCR amplification, linking a SUMO gene of fusion protein by using a Linker in front of a thaumatin promoter by using an overlap extension PCR method, respectively cutting a target protein expression sequence and pET9a plasmid by using Not I and EcoR I enzyme cutting sites, constructing escherichia coli expression plasmid by using T4 ligase, and transforming the plasmid into escherichia coli.

According to the technical scheme, the extraction of the recombinant thaumatin protein comprises the following steps: after the supernatant of the medium (100mL) was subjected to a needle treatment with 5mM sodium phosphate buffer (disodium phosphate 3.55g, sodium dihydrogen phosphate 3g in 5L water) at 4 ℃, the dialysate was passed through an SP-Sephadex C-25(Amersham biosciences) column (1.7 cm. times.5 cm) equilibrated with 5mM sodium phosphate buffer (pH7.0), recombinant thaumatin was eluted with a linear gradient of 0-500mM NaCl in 5mM sodium phosphate buffer (pH7.0), the protein fraction detected by SDS-PAGE was dialyzed against 5mM sodium phosphate buffer pH7.0, and the dialysate (12mL) was passed again through the SP-Sephadex C-25 column for further purification; bound proteins were eluted with a linear gradient of 20mM to 300mM NaCl in 5mM sodium phosphate buffer (pH 7.0); the recombinant thaumatin proteins were pooled and dialyzed against 5mM sodium phosphate buffer, pH 5.0.

In order to verify the beneficial effects of the invention, the following tests are specially carried out:

test 1. scavenging ability of anthocyanin mixture with beta-glucan for free radical

The hydroxyl radical is removed by Fenton reaction

Taking 3 colorimetric tubes with the volume of 10mL, respectively and sequentially adding 1.0mL7.5mmol/L ferrous sulfate iron solution, 1.0mL7.5mmol/L salicylic acid solution and 1.0mL7.5mmol/L hydrogen peroxide solution, sequentially adding 1.0mL anthocyanin and beta-glucan mixture into a No. 2 colorimetric tube, fixing the volume to the scale by using deionized water, heating in a water bath for 1h, sequentially adding 1.0mL anthocyanin into a No. 3 colorimetric tube, fixing the volume to the scale by using deionized water, and heating in a water bath for 1 h. The absorbance values were measured at 510nm wavelength and the clearance was calculated.

Clearance rate equation: clearance (%) ═ a₀-As)/A₀*100％

In the formula: a. the₀-no absorbance value of the test substance is added; as-absorbance after addition to assay

TABLE 1 statistics of radical scavenging by anthocyanin and beta-glucan mixtures

The test result shows that the mixture of the anthocyanin and the beta-glucan has good free radical scavenging capability and stronger free radical scavenging capability than that of the simple anthocyanin.

Experiment 2. experiment of the influence of the biscuit of the invention on the body weight of rats

After all rats were fed for 7 days at increased weight, they were randomly divided into 2 groups, a diet biscuit group (10) and a blank control group (10), respectively. The rats in the weight-reducing biscuit group are fed with weight-reducing biscuits, the rats in the blank control group are fed with conventional feed with the same weight, drinking water is freely carried out, the rats are irradiated for 12 hours/darkened for 12 hours, and the temperature is 23 +/-2 ℃. The weight of the feed is weighed after feeding for 28 days. Specific values are shown in table 1:

average body weight (g)	Day 0	7 days	14 days	28 days
					Weight-reducing biscuit group	375	361	347	326
Blank control group	381	378	369	367

TABLE 2 statistical table of rat body weights

As can be seen from Table 2, the weight of the rats fed with the diet biscuits is reduced remarkably, and the weight of the rats fed with conventional feed is not reduced remarkably, which shows that the diet biscuits have a good effect on improving the weight of obese rats.

It is noted that, herein, relational terms such as first and second, and the like may be used solely to distinguish one entity or action from another entity or action without necessarily requiring or implying any actual such relationship or order between such entities or actions. Also, the terms "comprises," "comprising," or any other variation thereof, are intended to cover a non-exclusive inclusion, such that a process, method, article, or apparatus that comprises a list of elements does not include only those elements but may include other elements not expressly listed or inherent to such process, method, article, or apparatus.

Finally, it should be noted that: although the present invention has been described in detail with reference to the foregoing embodiments, it will be apparent to those skilled in the art that changes may be made in the embodiments and/or equivalents thereof without departing from the spirit and scope of the invention. Any modification, equivalent replacement, or improvement made within the spirit and principle of the present invention should be included in the protection scope of the present invention.