CN113699017A

CN113699017A - Mulberry pomace vinegar and preparation method thereof

Info

Publication number: CN113699017A
Application number: CN202010997709.9A
Authority: CN
Inventors: 卢红梅; 陈莉; 杨双全; 吴煜樟
Original assignee: Guizhou University
Current assignee: Guizhou University
Priority date: 2020-09-21
Filing date: 2020-09-21
Publication date: 2021-11-26

Abstract

The invention relates to the field of food processing, in particular to mulberry pomace vinegar and a preparation method thereof. Adding sugar into mulberry pomace, uniformly stirring, adding yeast for fermentation, adding acetobacter after fermentation, adding frosted mulberry leaves for regulating water content to prepare vinegar mash, finishing fermentation when the total acid of the vinegar mash does not rise or slightly falls, spraying vinegar after fermentation, filtering, centrifuging, and sterilizing to obtain a finished product. The mulberry pomace vinegar is optimized in process, the optimal fermentation process conditions of the mulberry pomace vinegar are obtained, changes of indexes such as anthocyanin, acidity, polyphenol and flavone in the processes of alcoholic fermentation and acetic fermentation of fruit vinegar are monitored, and the fruit vinegar with high polyphenol and flavone content is developed through comprehensive analysis of physical and chemical indexes of finished fruit vinegar. The water content, the initial alcohol content and the fermentation temperature of the vinegar culture are accurate, so that the finished product can achieve the optimal fermentation effect, has good taste and optimal nutrition.

Description

Mulberry pomace vinegar and preparation method thereof

Technical Field

The invention relates to the field of food processing, in particular to mulberry pomace vinegar and a preparation method thereof.

Background

Mulberries (Mulberry), also known as mulberries, black mulberries and mulberries, change from green to red in the early maturation stage and from red to purple-black in the later stage, wherein the maturation stage is mainly 4-6 months. Mulberries mainly grow in regions with proper climates such as tropical and subtropical regions, mulberries are rich in anthocyanin, polysaccharide, polyphenol, amino acid, organic acid and other functional components, have the effects of tonifying liver and kidney, nourishing yin and supplementing blood, delaying senescence, regulating immunity and the like, most of byproducts such as pomace are not fully utilized in developing countries at present, and the discarded pomace can pollute the environment (Sethi B K, Nanda P K, Sahoo S. enhanced production of peptide by Aspergillus terreus NCFT 4269.10 usanb base peptides as substrate [ J ].3 Biotech,2016,6(1): 36.). However, China is one of the countries with the front fruit planting area and production place, and nearly millions of tons of fruit residues are discarded every year. Without proper treatment, the pomace resources are seriously wasted and even harmful to the environment (wisdom, etc. the utilization status and development trends of pomace [ J ] the feed industry, 2017,38(13): 44-46.). Therefore, it is very necessary to fully utilize the pomace. The mulberry pomace still has a large amount of soluble dietary fibers (Liaoli and the like. the extraction process of the soluble dietary fibers of the mulberry pomace is optimized [ J ]. Hubei agricultural science, 2014,53(24):6086-, therefore, not only is the environment pollution caused, but also the resource waste is caused, and according to research reports, a large amount of pomace is not fully utilized every year; therefore, the method has important research significance for comprehensive utilization of byproducts such as mulberry pomace and the like of enterprises.

Disclosure of Invention

Aiming at the problems, in order to solve the defects in the prior art, the invention aims to increase the added values of the pomace and the frost mulberry leaf by-products in Guizhou mulberry wineries, change waste into valuable and comprehensively utilize the mulberry pomace and the mulberry leaves. A Mori fructus pomace vinegar and its preparation method are provided.

The technical scheme of the invention is as follows:

a mulberry pomace vinegar is prepared by the following steps: blending mulberry pomace, adding saccharomycetes after blending, wherein the inoculation amount of the saccharomycetes is 0.04-0.05% of the total mass of the mulberry pomace, carrying out alcoholic fermentation at the temperature of 26-28 ℃, wherein the fermentation time is 5 days, adding acetobacter after fermentation, the inoculation amount of the acetobacter is 11.91-12.0% of the total mass of the mulberry pomace, adding frost mulberry leaves to adjust the water content to prepare vinegar fermented grains, carrying out vinegar pouring after fermentation, soaking for 7-9 hours, repeatedly soaking for three times, filtering, centrifuging and sterilizing to finally obtain a finished product.

The blending is to add mulberry juice and sugar into the mulberry pomace, wherein the adding amount of the sugar is 12-16% of the total mass of the mulberry pomace, and the adding amount of the mulberry juice is 21% of the total mass of the mulberry pomace.

Sugar can be added in the alcohol fermentation process, and the adding amount is the best 12-16% of the total mass of the mulberry pomace.

The water content of the vinegar mash is 79.44% -79.5% optimally.

The initial alcoholic strength is 7.63% vol-7.7% vol.

The fermentation temperature of the fermented grains of the vinegar is 32.1 ℃, and the fermented grains of the vinegar are stirred once every 24 hours.

The sterilization condition is carried out for 30min at 65 ℃; the centrifugation condition is 4000r/min, and the treatment time is 20 min.

A preparation method of mulberry pomace vinegar comprises the following steps: adding sugar into mulberry pomace, wherein the adding amount of the sugar is 12-16% of the total mass of the mulberry pomace, uniformly stirring, adding yeast, the inoculation amount of the yeast is 0.04-0.05% of the total mass of the mulberry pomace, performing alcohol fermentation at 26-28 ℃, wherein the fermentation time is 5 days, adding acetobacter after fermentation, the inoculation amount of the acetobacter is 11.91-12.0% of the total mass of the mulberry pomace, adding frost mulberry leaves, adjusting the water content to prepare vinegar fermented grains, pouring vinegar after fermentation, soaking for 7-9 hours, repeatedly soaking for three times, filtering, centrifuging, and sterilizing to finally obtain a finished product.

The main advantages of the invention are:

the invention optimizes the process of the mulberry pomace vinegar, monitors the change of indexes such as anthocyanin, acidity, polyphenol and flavone in the processes of alcoholic fermentation and acetic fermentation of fruit vinegar under the optimal fermentation process conditions of the mulberry pomace vinegar, and develops the fruit vinegar with higher polyphenol and flavone content through comprehensive analysis of physical and chemical indexes of finished fruit vinegar. The water content, the initial alcohol content and the fermentation temperature of the vinegar culture are accurate, so that the finished product can achieve the optimal fermentation effect, has good taste and optimal nutrition. Has good antioxidant and hypoglycemic effects and certain antibacterial function.

Drawings

FIG. 1-1 is a graph of response of moisture content versus alcohol content;

FIG. 1-2 is a contour plot of water content versus alcohol content;

FIG. 2-1 is a graph of water cut versus temperature response;

FIG. 2-2 is a contour plot of moisture content versus temperature;

FIG. 3-1 is a response plot of water content versus inoculum size;

FIG. 3-2 is a contour plot of water content versus inoculum size;

FIG. 4-1 is a graph of alcohol content versus temperature response;

FIG. 4-2 is a contour plot of alcohol content versus temperature;

FIG. 5-1 is a response surface diagram of alcohol content versus inoculum size;

FIG. 5-2 is a contour plot of alcohol content versus inoculum size;

FIG. 6-1 is a response surface plot of temperature versus inoculum size;

FIG. 6-2 is a contour plot of temperature versus inoculum size;

FIG. 7 Standard curve of reducing sugar content Figure 7 Standard curve of reducing sugar content;

FIG. 8 is a graph of the Standard curve Figure8 Standard curve of polyphenol content for polyphenol content;

FIG. 9 is a Standard curve Figure for flavone content, Figure 9 Standard curve for flavone content;

FIG. 10 shows the variation of alcohol and reducing sugar content in the fermentation process of Mulberry pomace alcohol, Figure10Changes in the content of alcohol and reducing sugar reducing the alcohol fermentation of mulberry pomace;

FIG. 11 shows the change of anthocyanin content in the alcoholic fermentation process of mulberry pomace, Figure 11Changes in the anti-pathological in content reduction of the mulberry deposit;

FIG. 12 is a graph showing The influence of different temperatures on The alcohol content of mulberry pomace during alcohol fermentation process, which shows The influence of fire 12 on The alcohol content of mulbery pomace;

FIG. 13 is a graph showing The effect of different temperatures on The anthocyanin content in The alcoholic fermentation process of mulberry pomace, Figure 13 The effect of differential temperature on The anti-bacterial content during The alcoholic fermentation process of mulberry pomace;

FIG. 14 is a graph showing The influence of different inoculation amounts on The alcohol content in The alcohol fermentation process of mulberry pomace, Figure 14The effect of differential inhibition on The alcohol content of mulbery pomace;

FIG. 15 shows the influence of different inoculation amounts of mulberry pomace on anthocyanin content in the alcoholic fermentation process, Figure 15 Effect of differential inoculation of mulum mulberry pore on the alcoholic fermentation content reduction alcohol fermentation;

FIG. 16 is a graph showing the Effect of different sucrose addition amounts on anthocyanin content in alcoholic fermentation of mulberry pomace, Figure 16 Effect of differential activities of sugar on the anti-cancer content in alcohol fermentation of mulberries;

FIG. 17 shows the variation of total acid content of fermented vinegar with different water contents, Figure 17 Changes in total acid content of vinegar with different water contents during the acetic acid fermentation process;

FIG. 18 shows the change of polyphenol content in vinegar grains with different water contents during the acetic fermentation process, Figure 18 Variation of polyphenol content of vitamin with different water content and acetic acid fermentation;

FIG. 19 shows the change of flavone content in vinegar grains with different water contents during fermentation process, Figure 19Changes in flavone content of vinegar grains with different water contents;

FIG. 20 shows the variation of total acid content in vinegar grains of different alcohol contents during the acetic fermentation process, Figure 20 Changes in total acid content of vinegar grains with different alcohol contents and with different alcohol content during the acetic fermentation process;

FIG. 21 shows the change of polyphenol content in vinegar grains with different alcohol contents during the acetic fermentation process, Figure 21 Changes in polyphenol content of vinegar grains with different alcohol contents and different amounts of acetic acid fermentation;

FIG. 22 shows the change of flavone content in vinegar grains of different alcohol contents during the acetic fermentation process, Figure 22 Changes in flavone content of vinegar with different alcohol contents and acetic acid fermentation;

FIG. 23 shows the variation of total acid content in the acetic acid fermentation process of vinegar grains fermented at different temperatures, Figure 23 Changes in total acid content of vitamin compensated at differential thermal treatment along with acetic acid fermentation;

FIG. 24 shows the change of polyphenol content in vinegar grains fermented at different temperatures during the acetic acid fermentation process, Figure24 Changes in polyphenol content of vitamin fermented at different temperatures;

FIG. 25 shows the change of flavone content in vinegar grains fermented at different temperatures during the acetic acid fermentation process, Figure 25 Changes in flavone content of vitamin fermented at different temperatures;

FIG. 26 shows that the total acid content of different amounts of fermented vinegar grains Changes by 26 Changes in total acid content of a vitamin with a second inoculation amount and decrease in acetic acid fermentation during the acetic acid fermentation process;

FIG. 27 shows the Variation of polyphenol content in different inoculated amount of vinegar grains during acetic acid fermentation, Figure 27 Variation of polyphenol content of vinegar grains with different inoculation amounts during acetic acid fermentation;

FIG. 28 shows the change of flavone content in vinegar grains with different inoculum sizes during the acetic acid fermentation process, Figure 28 Changes in flavone content of vinegar grains with different inoculum sizes

Detailed Description

The present invention is described in detail below by way of examples.

Example 1

1. Main instrument equipment

1.1, raw materials of mulberry pomace and frosted mulberry leaves: the Guizhou Tai and the Guizhou Tai are provided by the combined agriculture science and technology Limited liability company; BV818 wine yeast: purchased from Angel Yeast Ltd, stored in a refrigerator at 4 ℃ for later use; acetobacter pasteurianus: the Guizhou province fermentation engineering and the biological pharmacy key laboratory are preserved; sugar: white sugar of water-feeding well food factory in Kunming disc dragon area.

1.2 pretreatment of the raw and auxiliary materials

(1) Frosted mulberry leaves: cleaning fresh frost mulberry leaves picked in No. 11/month and No. 23 with water, naturally drying in a constant-temperature blast drying oven at 60 ℃, crushing to about 10 meshes, subpackaging in sealed bags, and storing in a refrigeration house at-15 ℃ for later use.

(2) Mulberry pomace: and (3) subpackaging the fresh mulberry pomace into a plurality of sterile sealed bags, and immediately storing the bags in a refrigerator at the temperature of-20 ℃ for later use.

1.3 Strain activation

(1) Yeast activation

BV818 active dry yeast was used for the experiments, the yeast activation procedure: weighing a certain amount of yeast, adding sugar water with the sugar content of 2% (5 times of the addition amount of the yeast), and performing water bath activation for 15-30 min at the temperature of 37 ℃.

(2) Preparation of Acetobacter pasteurianus

Solid-state culture: adding 1g of glucose, 2g of calcium carbonate, 2g of agar, 1g of yeast extract and 100mL of distilled water into a 500mL triangular flask, sterilizing the flask for 20min at 121 ℃ in an autoclave, cooling the sterilized culture medium to 50-60 ℃, adding 3mL of absolute ethyl alcohol, cooling the flat plate, re-streaking the preserved acetic acid bacteria, and placing the culture dish into a 30 ℃ constant-temperature incubator for culturing for 24-48 h.

Liquid fermentation culture: weighing 1% of glucose, 1% of yeast extract, 0.05% of magnesium sulfate and 0.05% of potassium dihydrogen phosphate, adding 100mL of distilled water to dissolve the culture medium, placing the dissolved culture medium in a 500mL triangular flask, sealing the flask mouth with a sealing film and newspaper, sterilizing the autoclave at 121 ℃ for 20min, cooling to 50-60 ℃ after the culture medium is sterilized, and then adding 3mL of absolute ethyl alcohol. Then inoculating the activated acetic acid bacteria strain into a liquid culture medium, and culturing for 48h in a shaking table at 30 ℃ and 150r/min for later use.

1.4 the process flow is as follows

A mulberry pomace vinegar is prepared by the following steps: mixing mulberry pomace, adding mulberry juice and sugar into 100kg of mulberry pomace, wherein the adding amount of the sugar is 15kg, and the adding amount of the mulberry juice is 21 kg.

Adding saccharomycetes after blending, wherein the inoculation amount of the saccharomycetes is 0.04kg, carrying out alcoholic fermentation at the temperature of 28 ℃, adding sugar during the fermentation process, the addition amount of the sugar is 15kg, the fermentation time is 5d, adding acetobacter after the fermentation, wherein the inoculation amount of the acetobacter is 11.91 kg of the total mass of the mulberry pomace, adding frost mulberry leaves, adjusting the water content to prepare vinegar fermented grains, carrying out stirring once at the temperature of 32.1 ℃, when the total acid of the vinegar fermented grains does not rise or slightly falls, finishing the fermentation, spraying vinegar after the fermentation, adding 120mL of sterilized distilled water, soaking for 8 hours, repeatedly soaking for three times, filtering, centrifuging for 4000r/min, 20min, and sterilizing for 30min at the temperature of 65 ℃, and finally obtaining a finished product.

2. Stage-by-stage sample detection and screening

2.1 test methods

2.1.1 sampling method

(1) Samples at the alcoholic fermentation stage: after the wine mash is uniformly stirred by a glass rod, respectively and uniformly weighing 20g of different samples, placing the samples in a 250mL triangular flask, adding 20mL of distilled water, soaking for 4h in a sealed manner, oscillating the samples once every half hour by using a vortex mixer, filtering the samples in a 50mL centrifugal tube by using filter paper, immediately storing the samples in a refrigerator at 4 ℃ in a sealed manner, and properly diluting a filtered sample liquid to determine indexes.

(2) Sample of acetic acid fermentation stage: sterilizing and cooling 3 stainless steel basins in an autoclave (121 ℃,20 min), pouring Vinegar grains into an iron basin, stirring uniformly, immediately weighing 20g of different samples, placing the samples into a 500mL triangular flask, adding 180mL of distilled water, soaking for 4h in a sealed manner, shaking once every half hour by using a vortex mixer, filtering the samples in a 50mL centrifuge tube by using filter paper, immediately storing the samples in a 4 ℃ refrigerator in a sealed manner, and properly diluting the filtered sample liquid to determine indexes (Xie X, Zheng Y, Liu X, et al.

2.1.2 determination of Alcoholic strength

The alcoholic content of the sample was measured with reference to the semi-micro distillation Method of AOAC Official Method 969.12 Alcohol in Wines By dichlorimate Oxidation (Zhangli. study of addition of yeast during fermentation of high-salt dilute soy sauce [ D ]. Guiyang: Guizhou university, 2019.).

2.1.3 determination of reducing sugars

[1] The reducing sugar in the sample is determined by referring to the research on determining the content of the reducing sugar by the colorimetric method of 3, 5-dinitrosalicylic acid [ J ] food science, 2008(08): 534-.

2.1.4 determination of anthocyanins

Reference (Giusti M M, Wrolstad R E. Characterisation and measurement of anticancer by UV-visible spectroscopy [ J ]. Current Protocols in Food Analytical Chemistry,2001.) slightly modified;

preparation of buffer solution at pH 1.0: accurately weighing 15g of KCl, diluting with distilled water and fixing the volume to 1000 mL; accurately measuring 17mL of concentrated hydrochloric acid, diluting to 1000mL with distilled water, and mixing a KCl solution and an HCl solution. The pH was adjusted with KCl solution (1.0. + -. 0.1).

preparation of pH4.5 buffer: accurately weighing 16.4gCH₃COONa was diluted with distilled water and made to 1000mL, and the pH was adjusted with HCl solution (4.5. + -. 0.1).

The determination step comprises: a1 mL sample (product of the alcoholic fermentation stage) was taken and made up to 10mL with distilled water. Transferring 2 parts of 1mL of sample solution, diluting to 10mL with buffer solutions with pH1.0 and pH4.5 respectively, shaking with an oscillator, standing in dark for 2h, replacing the sample with distilled water as blank control, and measuring absorbance at 510nm and 700 nm.

The anthocyanin content was calculated as follows:

in the formula: a ═ ph (a510nm-a700nm) 1.0- (a510nm-a700nm) ph4.5

MW-cyanidin-3-glucoside molecular weight, 449.2 g/mol;

DF-dilution factor;

ε -molar extinction coefficient, 26900L. mol^-1·cm^-1；

L is the number of centimeters of the optical path, 1 cm;

2.1.5 measurement of Polyphenol

Slightly modifying the reference (Lijing, Nioniaoyun, Wangxiao, and the like. Folin-Ciocalteus method for measuring the total polyphenol [ J ] in grapes and wine, China southern fruit tree, 2007(06): 86-87.);

(1) gallic acid monohydrate standard solution: accurately weighing 0.110 +/-0.001 g of monohydrate gallic acid, and dissolving the monohydrate gallic acid in a 100mL volumetric flask by using water, wherein the concentration of the monohydrate gallic acid in the solution is 1000 mg/L; using a 5mL graduated pipette to suck 0, 1.0, 2.0, 3.0, 4.0 and 5.0mL of the standard solution into a 100mL volumetric flask, and diluting with water to constant volume to obtain 0, 10, 20, 30, 40 and 50 mu g/mL of the gallic acid monohydrate standard solution respectively.

(2) Standard Curve preparation

And (3) preparing a standard curve: 1.0mL of gallic acid monohydrate standard solution was pipetted from 0, 10, 20, 30, 40 and 50. mu.g/mL series using a graduated pipette, and 5mL of water, 1mL of FC developer, and 3mL of 7.5% sodium carbonate solution were added to develop the gallic acid monohydrate in the amounts of 0, 1.0, 2.0, 3.0, 4.0 and 5.0. mu.g/mL, respectively. After standing for 2 hours, the absorbance of the series of standard solutions was measured at 765nm, a standard curve was plotted, and the equation was obtained.

(3) Experimental procedure

The sample (product of acetic acid fermentation stage) was diluted 120-fold, and then 1.0mL of the diluted sample solution was aspirated, and 5.0mL of water, 1mL of FC color developer, and 3mL of 7.5% sodium carbonate solution were added, respectively, to develop color. After standing for 2 hours, the absorbance of the sample was measured at 765nm and the concentration of polyphenol in the sample was calculated from the standard curve (equation).

2.1.6 measurement of flavones

The flavone content of the sample is detected by referring to the method specified in appendix C of GB/T19777-2013 geographic marking product Shanxi mature Vinegar.

2.1.7 determination of Total acids

And (3) measuring the total acid content in the sample by referring to an acid-base titration method in the measurement of the total acid in GB/T5009.41-2003 vinegar sanitary standard.

2.1.8 screening single factor of mulberry pomace vinegar alcohol fermentation

(1) Determination of alcoholic fermentation period

The method comprises the steps of dividing mulberry pomace into 18 parts, wherein 200g of mulberry pomace is divided, fermenting for 1d, 2d, 3d, 4d, 5d and 6d at 26 ℃ under the conditions that the adding amount of cane sugar and the adding amount of yeast are 14% and 0.03%, respectively measuring the change of sugar content and alcoholic strength in the alcohol fermentation process of the mulberry pomace, and finding out the optimal fermentation time.

(2) Influence of fermentation temperature on alcohol in alcohol fermentation process

The mulberry pomace is divided into 18 parts, each 200g, and under the conditions that the addition amount of sucrose is 14%, the addition amount of yeast is 0.03%, and the fermentation is carried out for 5d, six culture temperatures of 22 ℃, 24 ℃, 26 ℃, 28 ℃, 30 ℃ and 32 ℃ are tested to influence the alcohol fermentation, and the optimal temperature of the alcohol fermentation is discussed.

(3) Influence of yeast inoculation amount on alcohol fermentation process

The method comprises the steps of dividing mulberry pomace into 18 parts, each 200g of mulberry pomace, adding 14% of cane sugar, taking six inoculation amounts of 0.01%, 0.02%, 0.03%, 0.04%, 0.05% and 0.06% of yeast, measuring the alcoholic strength of the mulberry pomace at the fermentation temperature of 26 ℃, and discussing the optimal inoculation amount of the yeast during alcoholic fermentation at the fermentation time of 5 days.

(4) Influence of sucrose addition on alcohol fermentation process

The mulberry pomace is divided into 18 parts, each part is 200g, the adding amount of cane sugar of the pomace is adjusted to six levels of 10%, 12%, 14%, 16%, 18% and 20% according to different proportions, the fermentation temperature is 26 ℃, the yeast inoculation amount is 0.03%, the alcoholic strength of the mulberry pomace is measured during the 5 th fermentation period, and the optimal adding amount of cane sugar during the alcoholic fermentation is discussed.

2.2 screening of acetic acid fermentation single factor of mulberry pomace vinegar

A. Influence of water content on total acid in acetic acid fermentation process

Preparing 6 portions of mulberry pomace wine mash with the alcoholic strength of 7.5 vol%, respectively adding 10% acetic acid bacteria seed liquid, respectively adjusting the water content of the vinegar unstrained spirits to 60%, 65%, 70%, 75%, 80% and 85% by controlling the adding amount of frost mulberry leaves, placing the vinegar unstrained spirits in a constant-temperature incubator at 32 ℃ for fermentation, stirring once every 24 hours, immediately sampling and measuring the acidity after stirring uniformly, and when the total acid of the vinegar unstrained spirits does not rise or slightly fall, finishing the acetic acid fermentation, and discussing the optimal water content of the vinegar unstrained spirits fermentation.

B. Influence of alcoholic strength on total acid in acetic acid fermentation process

Uniformly dividing mulberry pomace into 6 parts, adjusting sugar degree, respectively adding 5.5% vol, 6.5% vol, 7.5% vol, 8.5% vol and 9.5% vol of alcohol content of wine mash into 10% acetic acid bacteria seed liquid, adding a proper amount of frost mulberry leaves to ensure that the water content of the vinegar unstrained spirit is 80%, then placing the vinegar unstrained spirit in a constant-temperature incubator at 32 ℃ for fermentation, stirring once every 24 hours, immediately sampling and measuring acidity, and when the total acid of the vinegar unstrained spirit does not rise or slightly falls, finishing acetic acid fermentation and discussing the optimal alcohol content of the vinegar unstrained spirit fermentation.

Adjusting the sugar degree: sucrose was supplemented with 1% vol alcohol based on 1.7g sugar, and the amount of addition was adjusted based on the different alcoholic strength requirements of acetic acid fermentation.

C. Influence of fermentation temperature on total acid in acetic acid fermentation process

Preparing mulberry pomace wine with the alcoholic strength of 7.5 vol%, adding 10% acetic acid bacteria seed liquid, adding a proper amount of frost mulberry leaves to enable the water content of the vinegar mash to be 80%, dividing the prepared vinegar mash into 6 parts, respectively putting the vinegar mash into constant-temperature incubators with the temperature of 28 ℃, 30 ℃,32 ℃, 34 ℃ and 36 ℃ for fermentation, stirring once every 24 hours, immediately sampling and measuring the acidity after stirring uniformly, and when the total acid of the vinegar mash does not rise or slightly fall, finishing the acetic acid fermentation, and discussing the optimal fermentation temperature of the vinegar mash fermentation. D. Influence of acetic acid bacterium inoculation amount on total acid in acetic acid fermentation process

Preparing 6 parts of mulberry pomace wine mash with the alcoholic strength of 7.5% vol, inoculating 8%, 10%, 12%, 14% and 16% of inoculation amount into acetic acid bacteria seed liquid, adding a proper amount of frost mulberry leaves to ensure that the water content of the vinegar mash is 80%, putting the vinegar mash into an incubator at 32 ℃, stirring once every 24 hours, immediately sampling and measuring the acidity after uniformly stirring, and when the total acid of the vinegar mash does not rise or slightly fall any more, finishing acetic acid fermentation, and discussing the optimal acetic acid bacteria inoculation amount of the vinegar mash fermentation.

2.3 response surface optimization test of acetic acid fermentation of mulberry pomace vinegar

And (3) selecting the water content, the alcoholic strength, the fermentation temperature and the inoculation amount of the vinegar grains according to a Box-Behnken design method by taking total acid as an investigation index through a single-factor test result to perform a four-factor three-horizontal response surface experiment, and determining the optimal fermentation condition. (see table one)

Watch 1

Data processing and analysis

The experimental data were processed using Excel2010, origin8.6 and Design-expert.v8.0.6 software, with 2 parallel experiments per treatment group.

3 results and analysis

3.1DNS Standard Curve plotting

Preparing glucose standard solutions with different concentrations, measuring the absorbance of the glucose standard solutions and drawing a reducing sugar content standard curve. The regression equation of the standard curve of the content of reducing sugar is that y is 0.4336x-0.0148, R²The linear relationship is good at 0.9991. (see FIG. 7)

3.2 drawing Standard Curve of Polyphenol content

Preparing gallic acid monohydrate standard solutions with different concentrations, and measuring the absorbance to draw a polyphenol standard curve. The regression equation of the standard curve of polyphenol content is that y is 0.0124x +0.0066, R²The linear relationship was good at 0.999. (see FIG. 8)

3.3 flavone content Standard Curve

Preparing anhydrous rutin standard solutions with different concentrations, and measuring the absorbance to draw a flavone standard curve. The regression equation of the standard curve of the flavone content is that y is 0.4421x +0.0034, R²The linear relationship is good at 0.9991. (see FIG. 9)

3.4 fermentation of Mulberry pomace Vinegar alcohol

3.4 influence of fermentation time on alcohol fermentation of Mulberry pomace

As shown in the results of fig. 10, the alcohol content of the morula mash tended to gradually increase and then become gentle as the fermentation time increased during the alcohol fermentation, and at the 5 th day, the alcohol content of the morula mash reached the maximum value of 7.71% vol, and at this time, the alcohol content was decreased slightly thereafter. And the content of reducing sugar is in a trend of rapidly reducing and then flatly reducing along with the fermentation time, and after the 4d, the content of reducing sugar in the mulberry pomace wine mash tends to a constant value, which indicates that the alcoholic fermentation is basically finished. During the fermentation, it was observed that a large number of bubbles were generated on the surface of the beer at fermentation 2d, gradually decreased at fermentation 4d, and were substantially absent on the surface of the beer at 5 d.

As shown in the results of fig. 11, the anthocyanin content tended to increase and then decrease with the increase of the fermentation time, and the anthocyanin content increased from 107.5mg/L to 115.5mg/L from the 1 st d to the 2 nd d, which may be attributed to the elution of anthocyanin from the raw material of mulberry pomace. When reaching the 6d, the content of the anthocyanin is reduced to 83.14mg/L, and is reduced by 28.02 percent on the basis of the maximum value of 115.5 mg/L. Probably because the anthocyanin content is easily influenced by factors such as illumination, sugar degree, ethanol and the like along with the prolonging of the fermentation time, the degradation of the anthocyanin occurs (Hongyu and the like, research on the change of bioactive substances and the antioxidant activity thereof in the whole residue fermentation process of mulberry fruit wine [ J ] food industry science and technology, 2015,36(23): 182-. And comprehensively selecting the fermentation time of 5d as a subsequent alcohol fermentation test from the influence of the fermentation time on the alcoholic strength and the anthocyanin.

3.6 influence of fermentation temperature on alcohol fermentation of Mulberry pomace

As can be seen from fig. 12, the alcoholic strength tended to increase first and then decrease as the fermentation temperature increased. The alcoholic strength reaches the maximum value of 7.88% vol at the temperature of 28 ℃, and the alcoholic strength is 7.37% vol at the temperature of 22 ℃, which is probably because yeast grows slowly, the activity of enzyme is low, the fermentation time is long, and the alcoholic strength is low due to the fact that the yeast is easily polluted by mixed bacteria or incomplete fermentation in the fermentation process. And when the temperature is higher than 28 ℃, the fermentation power is insufficient due to the rapid growth of yeast, and cells are easy to age and autolyze (Wuqingyuan, highland barley vinegar fermentation process and characteristic research thereof [ D ]. Chongqing: southwest university, 2017.).

As can be seen from fig. 13, the anthocyanin content tended to decrease with increasing temperature during the alcoholic fermentation of mulberry pomace. The content of anthocyanin is reduced to 87.34mg/L, 85.34mg/L and 84.01mg/L at 28 deg.C, 30 deg.C and 32 deg.C, respectively. This is probably because anthocyanin belongs to heat-sensitive substance, and the decomposition rate increases with an increase in temperature, thereby decreasing the anthocyanin content. Comprehensively selecting the fermentation temperature of 26-28 ℃ from the influence of the temperature on the alcoholic strength and the anthocyanin as a subsequent alcoholic fermentation test.

3.8 influence of Yeast inoculum size on alcohol fermentation of Mulberry pomace

As can be seen from fig. 14, the alcohol content tended to increase first and then decrease slightly with increasing yeast inoculum size, and the alcohol content of the mash reached the highest value at 0.05% yeast addition. When the yeast inoculation amount is less than 0.04%, the alcohol content of the mulberry pomace wine mash is low, which indicates that the sucrose is not fully utilized in the alcohol fermentation process. When the inoculation amount is higher than 0.06%, the alcohol content of the mulberry pomace wine mash is slightly lower, and the too large inoculation amount can cause quick yeast aging and low alcohol fermentation capacity.

[1] As can be seen from fig. 15, the anthocyanin content of the mulberry fruit residues tended to decrease during the alcoholic fermentation process as the yeast inoculation amount increased. When the inoculation amount of the yeast is 0.04%, the anthocyanin content is 85.32mg/L and has little difference from the anthocyanin content of 0.05% and 0.06%. The reason for the decrease of anthocyanin with the increase of the inoculation amount is probably that yeast degrades the glycoside form of anthocyanin to generate anthocyanin aglycone or polymerizes to be polymeric anthocyanin, which leads to the decrease of water-soluble anthocyanin. Yeast can release secondary metabolites such as pyruvic acid, acetaldehyde, etc. during fermentation and react with anthocyanin to produce some macromolecular derivatives (Liu Zhi et al. dynamic changes of main and functional components during ethanol fermentation of mulberry juice [ J ] food and fermentation industry, 2006(12): 138-. Therefore, the influence of the yeast inoculation amount on the alcoholic strength and anthocyanin is comprehensively selected, and the inoculation amount of 0.04-0.05% is used as a subsequent alcoholic fermentation test.

As can be seen from fig. 16, the content of anthocyanin in the mulberry pomace tended to decrease as the amount of sucrose added increased during the alcoholic fermentation. After the alcoholic fermentation is finished, when the adding amount of sucrose is 10%, 12%, 14%, 16%, 18% and 20%, the corresponding anthocyanin content is 89.64 mg/L, 89.3mg/L, 88.47mg/L, 87.47mg/L, 86.96mg/L and 85.29mg/L respectively, and the influence of different adding amounts of sucrose on the anthocyanin content of mulberry pomace is not greatly different. Therefore, the influence of the addition amount of the sucrose on the alcoholic strength and the anthocyanin is comprehensively considered, and the addition amount of the sucrose is 12-16% and is used as a subsequent alcoholic fermentation test.

3.12 acetic fermentation of Mulberry pomace Vinegar

Influence of water content on total acid in acetic acid fermentation process

As can be seen from fig. 17, in the acetic acid fermentation stage of 14 days, the total acid content of the fermented vinegar grains with different water contents tended to increase slowly and then decrease. The total acid of the vinegar residue of the mulberry residue vinegar is the highest value when the water content of the vinegar residue is 80%, and the total acid of the vinegar residue is increased from 0.605g/100g of the initial value to 4.85g/100 g. When the water content is less than 80%, the lower the water content, the lower the total acid content of the fermented vinegar. After the acetic acid fermentation is finished, the total acid of the vinegar grains with the water content of 60%, 65%, 70% and 75% is respectively

From the initial value, to 1.23g/100g, 1.926g/100g, 2.987g/100g and 4.07 g/100 g. The reason may be that when the water content of the raw vinegar mash of the mulberry pomace is too low, the interiors of the frost mulberry leaves and the mulberry pomace are too loose, the dissolved oxygen is large, and acetic acid bacteria utilize a large amount of nutrients to metabolize, so that the acetic acid bacteria age, the alcohol utilization is incomplete, and the total acid content of the raw vinegar mash is low. When the water content of the vinegar grains is 85%, the total acid is 4.003g/100g, the water content is too high, the dissolved oxygen is not high, and the content of acetic acid bacteria metabolites is low. Some studies show that (Zhuyaodi Zhenjiang aromatic vinegar)

The intelligent online monitoring and distribution research of solid-state fermentation parameters [ D ] Zhenju university, 2016.) shows that the water content of the fermented vinegar in the solid-state fermentation vinegar is about 60-70%, and the research shows that the fermented vinegar with the water content of 80% has better fermentation acid yield and less accumulated liquid, probably because the water retention rates of mulberry pomace and frosted mulberry leaves are higher, so that the water content of the fermented vinegar is increased.

As can be seen from fig. 18, as the fermentation time was prolonged, the content of polyphenols in the fermented vinegar grains having different water contents tended to decrease during the acetic acid fermentation process. When the total acid of the vinegar grains with the water content of 60%, 65%, 70% and 80% reaches the maximum value in 11d, the polyphenol content is respectively reduced by 49.97%, 57.17%, 55.77% and 52.64% from the initial value of 0.643g/100g, 0.645g/100g, 0.651g/100g and 0.622g/100 g; while the water content was 75% and 85% with the highest total acid at 10d and 12d, respectively, and the polyphenol content was reduced from the initial values of 0.630g/100g and 0.594g/100g by 55.27%, 53.15%, respectively. Therefore, the change range of the polyphenol content with the water content of 60 percent is minimum, and the water content is 80 percent. As can be seen from FIG. 19, in the acetic acid fermentation stage of 1-14 days, the content of flavone in the vinegar substrate with different water content tends to decrease during the acetic acid fermentation process. At the end of acetic fermentation, the flavone content of the vinegar grains with the water content of 60%, 65%, 70%, 75%, 80% and 85% is respectively reduced by 34.48%, 45.59%, 39.99%, 36.84%, 45.21% and 40.42% from the initial value of 0.394g/100g, 0.399g/100g, 0.396g/100g, 0.390g/100g, 0.451g/100g and 0.490g/100 g. The three indexes of total acid, polyphenol and flavone are comprehensively considered, and the water content of the vinegar grains at the later stage is selected from 75%, 80% and 85% for carrying out optimization test.

As can be seen from FIGS. 21 and 22, in the whole acetic fermentation process, along with the extension of the fermentation time, the contents of polyphenol and flavone in the vinegar substrate tend to decrease, and at 0-6 d, the decrease rate of the polyphenol and flavone is higher, and the speed is gradually gentle at the later stage of the fermentation. After the acetic acid fermentation is finished, the initial alcoholic strength of the fermented vinegar grains is 5.5% vol, 6.5% vol, 7.5% vol, 8.5% vol and 9.5% vol, the polyphenol content is respectively reduced from the initial values of 0.607g/100g, 0.608g/100g, 0.606g/100g, 0.602g/100g and 0.599g to 0.297g/100g, 0.287g/100g, 0.271g/100g, 0.278 g/100g and 0.310g/100g, and the flavone content of the fermented vinegar grains is respectively reduced from the initial values of 0.450g/100g, 0.444g/100g, 0.448g/100g, 0.446g/100g and 0.449g/100g to 0.244 g/100g, 0.235g/100g, 0.230g/100g, 0.229g/100g and 0.256g/100 g. The three indexes of total acid, polyphenol and flavone are comprehensively considered, and the initial alcoholic strength of the vinegar grains at the later stage is optimized by selecting 6.5 percent vol, 7.5 percent vol and 8.5 percent vol.

3.18 Effect of fermentation temperature on Total acid in the acetic acid fermentation Process

From the results of FIG. 23, it is seen that the total acid content in the acetic acid fermentation process tends to increase first and then decrease as the fermentation temperature increases. And (4) when acetic acid is fermented for the 11d, the total acid of the mulberry pomace vinegar mash at the temperature of 32 ℃ reaches the maximum value, and the content of the total acid is 4.84g/100 g. When the fermentation temperature of the vinegar grains is too low, the growth and metabolism of acetic acid bacteria are not facilitated, and the fermentation time is prolonged. When the temperature is 28 ℃, the acid production speed of the vinegar grains in the early stage of fermentation is slow, the utilization rate of substrates such as ethanol is low, and the acid production of the fruit vinegar fermented at the temperature is low; when the fermentation temperature is 36 ℃, the aging of the acetic acid bacteria can be accelerated to weaken the acid production capability by too high temperature; and the volatilization of acetic acid generated in the vinegar grains is accelerated, so that the total acid content is reduced.

From the results of FIG. 24, it was found that the polyphenol content of the fermented vinegar showed a sharp and gradual decrease with the time of fermentation under different temperature conditions. The higher the temperature is, the faster the polyphenol content in the vinegar culture is reduced. After the fermentation is finished, the polyphenol content of the vinegar grains at the temperature of 28 ℃, 30 ℃,32 ℃, 34 ℃ and 36 ℃ is respectively reduced by 51.65%, 53.94%, 54.86%, 60.64% and 66.12% from the initial value of 0.605g/100g, 0.606g/100g, 0.602g/100g, 0.608g/100g and 0.612g/100 g. The fermentation temperature of 28 ℃ has the least influence on the polyphenol content, but the fermentation time is long and the alcohol conversion rate is reduced. From the results of FIG. 25, it was found that the higher the temperature was, the faster the flavone content in the fermented vinegar was decreased. When the time is 0-6 days, the flavone content is reduced at a high speed, and the change is gentle within 7-14 days. After the acetic fermentation is finished, the flavone content of the vinegar grains at the temperature of 28 ℃, 30 ℃,32 ℃, 34 ℃ and 36 ℃ is respectively reduced by 46.84%, 48.88%, 50.36%, 54.47% and 57.01% compared with the initial value of 0.46g/100g, wherein the difference between 30 ℃ and 32 ℃ is not large. Therefore, the fermentation temperature of 32 ℃ has a good effect in consideration of the integration of three indexes of total acid, polyphenol and flavone, so that response surface optimization tests are carried out at the later stage by using 30 ℃,32 ℃ and 34 ℃.

3.21 Effect of acetic acid inoculum size on Total acid in acetic acid fermentation Process

As can be seen from FIG. 26, the total acid tended to increase and then decrease with increasing inoculation amount during the acetic acid fermentation. It can be obviously seen that the larger the inoculation amount of the acetic acid bacteria is, the larger the acid production rate is in the early stage of fermentation. And in the 9 th day of acetic fermentation, the total acid content of the vinegar grains with the inoculation amount of 16% reaches 4.06g/100g, but the alcohol content is diluted too much due to the overlarge inoculation amount of the acetic acid bacteria, so that the acid yield in the vinegar grains is greatly reduced. When the inoculation amount is 8%, the fermentation time is too long, the acid production speed is slow, and the dominant bacteria do not fully utilize the fermentation substrate in the fermentation process, so that the total acid content of the vinegar grains is not high. At 11d, the total acid content of the vinegar residue with the inoculation amount of 12% reaches a maximum value of 5.01g/100g, the inoculation amount is 10% times, and the total acid content is 4.855g/100 g.

As can be seen from fig. 27, the polyphenol content of the fermented vinegar grains tended to decrease with the fermentation time during the entire acetic acid fermentation period. In 1 st to 8 th days, the reduction rate of the polyphenol content is high, and the reduction rate of later fermentation is gentle. The initial contents of polyphenol of the vinegar grains with the inoculation amounts of 8%, 10%, 12%, 14% and 16% are respectively 0.636g/100g, 0.62g/100g, 0.612g/100g, 0.613 g/100g and 0.610g/100 g; after the acetic fermentation is finished, the polyphenol content of the vinegar grains with the inoculation amount of 8%, 10%, 12%, 14% and 16% is respectively reduced by 55.78%, 54.05%, 54.15%, 55.15% and 56.44%. The reduction range of the polyphenol content in8 percent of vinegar grains is large, the reduction range of the polyphenol content is large due to the factors of small inoculation amount, long fermentation time, temperature and the like. As can be seen from FIG. 28, the flavone content in the fermented vinegar grains tended to decrease with the fermentation time, and the difference between the flavone contents of the fermented vinegar grains with different inoculation amounts was small. The initial contents of flavones of the vinegar culture with the inoculation amounts of 8%, 10%, 12%, 14% and 16% are respectively 0.458g/100g, 0.457g/100g, 0.438g/100g, 0.436g/100g and 0.441g/100g, and the contents of flavones of the vinegar culture with the inoculation amounts of 8%, 10%, 12%, 14% and 16% are respectively reduced by 47.36%, 48.87%, 48.35%, 49.63% and 49.77% compared with the initial values when the acetic acid fermentation is finished. Therefore, considering the combination of three indexes of total acid, polyphenol and flavone, the fermentation effect is the best when the inoculation amount is 12%, and therefore 10%, 12% and 14% are used for response surface optimization tests in the later period.

3.4 Mulberry pomace vinegar acetic acid fermentation response surface test optimization

3.4.1 response surface Experimental results and analysis of variance

The test results of the acetic acid fermentation response surface of the mulberry pomace are shown in table 3.4, and the analysis results of the variance are shown in table 3.5.

TABLE 3.4 response surface design and results

Table 3.4 Design and results of response surface experiment

TABLE 3.4

And (3) performing regression analysis on the data by adopting Design-expert.V8.0.6 software to obtain an analysis of variance table of a regression equation, wherein a quadratic regression equation is obtained as shown in a table 3.5:

Y＝+5.01-0.11A+0.17B+0.020C-0.058D+0.12AB+8.750E-003AC-0. 065AD+0.039BC+4.500E-003BD-0.13CD-0.42A²-0.60B²-0.39C²-0.63D²

TABLE 3.5 analysis of variance of regression equation

Table 3.5 Analysis of variance of regression equation

TABLE 3.5

Note: represents a 0.05 significant level; represents 0.01 significant level

From the analysis of variance of the regression equation in Table 3.5, the model P<0.0001, the response surface model is extremely remarkable; mismatching error P-0.1556>0.05, not significant at α ═ 0.05 levels. Determining the coefficient R²0.9796, the correction of the model determines the coefficient R² _Adj0.9593, the mathematical model can be used for predicting the response value of the process condition of the mulberry pomace vinegar. From the polynomial regression model equation of the response values, the influence of A and B of the primary factor on the total acid is very significant (P)<0.01), factor D affects significantly (P)<0.05) Factor C did not significantly affect (P)>0.05), and the factors influencing the acetic acid content of the mulberry pomace vinegar are respectively B>A>D>C, i.e. alcohol content>Water content>Amount of inoculation>And (4) fermentation temperature. 4 factors P of model quadratic term<0.0001, and the influence on the total acid is extremely remarkable (P)<0.01). The interaction items of the model are extremely significant in CD, significant in AB, and insignificant in AC, AD, BC and BD (P)>0.05)。

3.4.2 response surface analysis

The influence of interaction among all factors on the total acetic acid of the mulberry pomace can be visually seen through the response curved surface and the contour map, and the interaction map and the contour map are shown in figures 1-5. FIG. 1 shows the interaction between water content and alcohol content, including FIGS. 1-1 and 1-2, with steeper slopes of the Response surface, indicating that these two factors interact significantly with total acid. When the water content of the vinegar grains is 77-81% and the alcoholic strength is 7-8% vol, the total acid content is close to the highest point, and the acid production capacity is strong in the range. The axial contour plot of alcohol content is denser than the water content, indicating that alcohol content has a greater impact on total acid than the water content of the raw material. The contour line is elliptical, which shows that the interaction of the two factors is strong and the influence is obvious. As can be seen in FIG. 2, the Response curve of water content versus temperature is steeper, indicating that these two factors interact significantly with the Response value (total acid content). From the contour map, it is known that the axial contour line of the water content is denser than the fermentation temperature, which indicates that the water content of the raw material has a larger influence on the total acid than the fermentation temperature, and the contour line is circular, which indicates that the interaction of the two factors is weak and the influence is not significant.

As shown in FIG. 3, the Response curve of water content and inoculation amount is steeper, indicating that the interaction of the water content and the inoculation amount of the vinegar mash with the total acid is more significant. The axial contour lines of the water content are denser than the inoculation amount, which shows that the water content of the vinegar grains has larger influence on the total acid than the inoculation amount, and the contour lines are circular, which shows that the interaction of the two factors is weaker and the influence is not obvious.

As can be seen in FIGS. 4, the Response curves for alcohol and temperature are steeper, indicating that both factors are more sensitive to total acid interaction. When the alcoholic strength is 7-8 vol% and the water content of the vinegar grains is 77-81%, the axial contour lines of the alcoholic strength are denser from the contour lines, which shows that the influence of the alcoholic strength on total acid is large compared with the temperature, and the contour lines are circular, which shows that the interaction of the two factors is weak and the influence is not obvious.

As can be seen from the results of the Response surface and surface maps of alcohol and vaccination volume in FIG. 5 (including FIGS. 5-1 and 5-2), the Response curves for alcohol content and inoculum size are steeper, indicating that these two factors interact significantly with total acid. Compared with the inoculation amount, the contour lines along the axial direction of the alcoholic strength are more dense, which shows that the alcoholic strength has larger influence on total acid than the inoculation amount, and the contour lines are circular, which shows that the interaction of the two factors is weaker and the influence is not obvious.

As can be seen in FIG. 6 (including FIGS. 6-1 and 6-2), the response curves for temperature and inoculum size are steeper, indicating that both factors of temperature and inoculum size interact significantly with total acid. When the temperature is 31-33 ℃ and the inoculation amount is 11-13%, the method is beneficial to acetic acid fermentation and acid production. Compared with the temperature, the density is higher along the axial contour line of the inoculation amount, which shows that the influence of the inoculation amount on the total acid is larger than the temperature, and the contour line is elliptic, which shows that the interaction of the two factors is stronger and the influence is obvious.

4 verification test

The optimal process conditions for acetic acid fermentation of mulberry pomace obtained by analysis and optimization of software Design-expert.V8.0.6 are as follows: the water content of the vinegar mash is 79.44%, the initial alcoholic strength is 7.63% vol, the fermentation temperature is 32.08 ℃, and the inoculation amount of acetic acid bacteria is 11.91%. In order to check the consistency of the prediction result and the real situation, the optimization conditions are subjected to verification experiments. But in consideration of actual operation conditions, the optimal fermentation process conditions are corrected to that the water content of the fermented grains of the vinegar is 79.5%, the initial alcoholic strength is 7.7% vol, the fermentation temperature is 32.1 ℃, the inoculation amount of the acetic acid bacteria is 12.0%, the average value of the total acid in the fermented grains of the vinegar is 5.02g/100g through verification experiments, the relative error between the predicted value of the total acid of the fermented grains of the vinegar and the predicted value of the total acid of the fermented grains of the vinegar is 5.03g/100g is small, the accuracy of the experiment result is good, and the response surface method is proved to be feasible for optimizing the acetic acid fermentation process of the mulberry pomace vinegar.

5, test results show that: comprehensively considering the influence on the alcoholic strength and anthocyanin, wherein the fermentation time is 5 days, the yeast inoculation amount is 0.04-0.05%, the fermentation temperature is 26-28 ℃, the mulberry pomace alcoholic fermentation effect is good, and in order to ensure the normal operation of the subsequent acetic acid fermentation, the addition amount of sugar is 12-16% to be used as a subsequent alcoholic fermentation test.

According to the single-factor test and the Box-Benhnken response surface optimization test, the regression model of the quadratic polynomial of the mulberry pomace vinegar is as follows:

Y＝+5.01-0.11A+0.17B+0.020C-0.058D+0.12AB+8.750E-003AC-0.065 AD+0.039BC+4.500E-003BD-0.13CD-0.42A²-0.60B²-0.39C²-0.63D²the optimal fermentation process conditions of the mulberry pomace vinegar obtained through the response surface test are as follows: the water content of the vinegar mash is 79.5%, the initial alcoholic strength is 7.7% vol, the fermentation temperature is 32.1 ℃, the inoculation amount of acetic acid bacteria is 12.0%, and under the condition, the average value of total acid in the vinegar mash is 5.02g/100 g. The relative error of the theoretical value of the total acid of the fermented grains of vinegar is smaller, which indicates that the response surface method is feasible for optimizing the acetic acid fermentation process of the mulberry pomace vinegar.

Claims

1. The mulberry pomace vinegar is characterized in that: the preparation method comprises the following steps: blending mulberry pomace, adding saccharomycetes after blending, wherein the inoculation amount of the saccharomycetes is 0.04-0.05% of the total mass of the mulberry pomace, carrying out alcoholic fermentation at the temperature of 26-28 ℃, wherein the fermentation time is 5 days, adding acetobacter after fermentation, the inoculation amount of the acetobacter is 11.91-12.0% of the total mass of the mulberry pomace, adding frost mulberry leaves to adjust the water content to prepare vinegar fermented grains, carrying out vinegar pouring after fermentation, soaking for 7-9 hours, repeatedly soaking for three times, filtering, centrifuging and sterilizing to finally obtain a finished product.

2. The mulberry pomace vinegar according to claim 1, wherein: the blending is to add mulberry juice and sugar into the mulberry pomace, wherein the adding amount of the sugar is 12-16% of the total mass of the mulberry pomace, and the adding amount of the mulberry juice is 21% of the total mass of the mulberry pomace.

3. The mulberry pomace vinegar according to claim 1, wherein: sugar can be added in the alcohol fermentation process, and the adding amount is the best 12-16% of the total mass of the mulberry pomace.

4. The mulberry pomace vinegar according to claim 1, wherein: the water content of the vinegar mash is 79.44% -79.5% optimally.

5. The mulberry pomace vinegar according to claim 1, wherein: the initial alcoholic strength is 7.63% vol-7.7% vol.

6. The mulberry pomace vinegar according to claim 1, wherein: the fermentation temperature of the fermented grains of the vinegar is 32.1 ℃, and the fermented grains of the vinegar are stirred once every 24 hours.

7. The mulberry pomace vinegar according to claim 1, wherein: the sterilization condition is carried out for 30min at 65 ℃; the centrifugation condition is 4000r/min, and the treatment time is 20 min.

8. A method for preparing the mulberry pomace vinegar of claim 1, which is characterized by comprising the following steps: the preparation method comprises the following steps: adding sugar into mulberry pomace, wherein the adding amount of the sugar is 12-16% of the total mass of the mulberry pomace, uniformly stirring, adding yeast, the inoculation amount of the yeast is 0.04-0.05% of the total mass of the mulberry pomace, performing alcohol fermentation at 26-28 ℃, wherein the fermentation time is 5 days, adding acetobacter after fermentation, the inoculation amount of the acetobacter is 11.91-12.0% of the total mass of the mulberry pomace, adding frost mulberry leaves, adjusting the water content to prepare vinegar fermented grains, pouring vinegar after fermentation, soaking for 7-9 hours, repeatedly soaking for three times, filtering, centrifuging, and sterilizing to finally obtain a finished product.