CN105349690B - method for monitoring fermentation process of solid-state brewed vinegar based on acid production index of microbial community - Google Patents
method for monitoring fermentation process of solid-state brewed vinegar based on acid production index of microbial community Download PDFInfo
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Abstract
The invention belongs to the field of biological fermentation, and particularly relates to a method for monitoring a fermentation process of solid-state brewed vinegar based on an acid production index of a microbial community. In order to overcome the defects of the traditional monitoring method for the fermentation process of brewed vinegar, the invention adopts a high-throughput sequencing technology to measure the microbial community structure of the fermented grains in the solid fermentation process of the vinegar, then calculates the acid production index of the microbial community by utilizing the relative abundance value of microorganisms related to the formation of total acid, substitutes the total acid prediction equation of the fermented grains to calculate the total acid content prediction value of the fermented grains sample, can know whether the fermentation process of the batch is faster or slower after being compared with the standard change curve of the total acid of the fermented grains, can timely adjust the fermentation process parameters according to the result, and timely judges and predicts the fermentation ending time.
Description
Technical Field
The invention belongs to the field of biological fermentation, and particularly relates to a method for monitoring a fermentation process of solid-state brewed vinegar based on an acid production index of a microbial community.
background
the production history of vinegar in China is long, the brewing process is a multi-strain mixed fermentation process, and vinegar with full flavor and soft sourness is produced through the metabolism of various microorganisms. There are two main fermentation stages in the production of vinegar: alcoholic fermentation and acetic fermentation. In the alcohol fermentation stage, starchy materials such as glutinous rice, sorghum and the like are subjected to a series of metabolism of mould and yeast to finally generate alcohol; the key step of determining the flavor and quality of the vinegar in the acetic acid fermentation stage is that a plurality of microorganisms participate in the fermentation together to convert ethanol into a plurality of flavor substances such as acids and the like, so that the microbial flora is the guarantee of the flavor and quality of the solid-state brewed vinegar product.
At present, vinegar fermentation process control of numerous vinegar manufacturers in China is mainly based on traditional operation experience, operation is mainly carried out according to a fixed fermentation method and fermentation time, temperature and flavor sense are detected only in the fermentation process, and the judgment of the end point of the solid-state acetic acid fermentation process depends on physical and chemical indexes such as total acid content, non-volatile acid content and other metabolites, so that the controllability of the whole solid-state acetic acid fermentation process is not high. Total acid, non-volatile acid, volatile flavor substances and the like in the vinegar grains are all results of metabolic activities of vinegar-making microbial floras, so that the tracking of the solid fermentation process of the vinegar by metabolite indexes such as the total acid and the like often has hysteresis, and once the total acid and the non-volatile acid are found to be low in content, the timely adjustment of the process in the fermentation process is difficult to carry out, so that the batch difference of the product quality is caused, and particularly the phenomenon that the product flavor difference is large occurs. In addition, the total acid, the non-volatile acid, the flavor substances and the like are all metabolites of the microorganisms, and compared with the change process of the microorganisms, the fermentation process has hysteresis.
With the rapid development of the microbial community high-throughput sequencing technology, people can rapidly research the species, the quantity and the functions of microorganisms in a specific environment from a gene level, which is greatly helpful for people to know the information of the complex microbial environment. Many researchers at home and abroad apply the high-throughput sequencing technology to the micro-ecological research on prokaryotes and eukaryotes in systems such as excrement, soil, seabed sludge, sewage and the like, and also have very wide application in the field of traditional fermented foods, wherein the research on the composition and distribution of microorganisms of Mexico fermented corn balls, Japanese black vinegar, Korean pickles, cheese Italian sausages and the like obtains a compelling result, provides a valuable theoretical basis for better understanding of the mechanisms of traditional fermentation at home and abroad, and provides theoretical data support for the reform and progress of the traditional fermentation industry.
The invention provides a monitoring method for a fermentation process of solid-state brewed vinegar based on an acid production index of a microbial community. The method adopts a high-throughput sequencing technology to measure the microbial community structure of the vinegar culture in the middle of the solid-state fermentation process, then calculates the acid production index of the microbial community by using the relative abundance value of microorganisms related to the formation of total acid, substitutes the total acid prediction equation of the vinegar culture to calculate the total acid content prediction value of the vinegar culture sample, can know whether the fermentation process of a batch is faster or slower after being compared with the standard change curve of the total acid of the vinegar culture, can timely adjust the fermentation process parameters according to the result, and timely judges and predicts the fermentation ending time. .
disclosure of Invention
The invention aims to solve the defects of controllability and unstable batch quality of the conventional vinegar solid-state brewing process, provides a method for monitoring the normal operation of vinegar solid-state fermentation by using a microbial community acid production index from a microbial acid production mechanism, has high accuracy and lays a theoretical foundation for the conversion from empirical control to scientific control in the solid-state vinegar brewing industry in China.
The invention is realized by the following scheme: sampling a vinegar culture sample in the fermentation process of solid-state fermentation vinegar, extracting total DNA of a microbial community in the vinegar culture sample, performing high-throughput sequencing on a V1-V3 region of a genetic development marker 16S rDNA of a sample genome through a high-throughput sequencing technology, substituting a microbial relative abundance value which is closely related to total acid of the vinegar culture in the vinegar culture microbial community into a microbial community acid production index calculation equation, and substituting the calculated acid production index into a vinegar culture total acid content prediction equation, thereby obtaining a prediction value of the total acid content of the vinegar culture. And comparing the predicted value of the total acid of the fermented grains with the variation standard curve of the total acid of the fermented grains, so that the actual progress of the batch of solid-state fermented vinegar can be judged and predicted, and the adjustment of the fermentation parameters of the vinegar can be guided. The method monitors the vinegar brewing process by monitoring the abundance of microorganisms related to acid production in vinegar grains, has high accuracy in acid production prediction and good monitoring effect on controlling the quality of vinegar, and has the following specific implementation route:
(1) extracting total DNA of the microbial community of the vinegar culture: sampling 100g of a vinegar culture sample from a vinegar factory, fully and uniformly mixing the sample in a sterile plastic package bag, taking 5g of the vinegar culture, and extracting total DNA of a microbial community in the vinegar culture through the steps of liquid nitrogen grinding, lysozyme treatment, chloroform-isoamylol extraction, ethanol washing, RNaseA digestion and the like.
(2) High-throughput sequencing analysis of the microbial community of the vinegar culture: amplifying the sequence of the 16S rRNA gene V1-V3 region in the total DNA of the microbial community, and purifying and quantifying the amplified product. And (3) after all amplification products are mixed in equal proportion, performing high-throughput sequencing on a sequencer, wherein the average length of the sequencing products is 500 bp. The relative abundance of each type of microorganism in the entire microbial community was obtained after the sequencing analysis was completed.
(3) Calculation of acid production index of microbial community: substituting the relative abundance of microorganisms (Acetobacter, Xanthomonas, Sphingomonas, Rhizobium, Pantoea, Methylobacterium and Staphylococcus) which form positive correlation with the total acid of the vinegar culture in the microbial community of the vinegar culture into a microbial acid production index calculation equation to obtain the microbial community acid production index.
(4) Predicting the total acid content of the vinegar grains: and (4) substituting the acid production index of the microbial community obtained by calculation in the step (3) into a total acid content prediction equation of the fermented vinegar substrate, so that the total acid content of the fermented vinegar substrate can be predicted according to the relative abundance of the microbes.
(5) Judging the fermentation process of the solid-state brewed vinegar: and (4) comparing the predicted value of the total acid of the fermented grains with the standard curve of the total acid change of the fermented grains, so as to judge and predict the actual progress of the batch of solid-state fermented vinegar and guide the adjustment of the vinegar fermentation parameters.
The invention provides a method for monitoring the fermentation process of vinegar solid state fermentation based on a microbial community acid production index. The fermentation trend can be predicted by determining the relative abundance of microbial communities and calculating the acid production index, so that the method is beneficial to adjusting process parameters in time and judging the fermentation finishing time in advance.
Description of the drawings:
FIG. 1 sequence length distribution of DNA pyrophosphate sequencing of microbial community of solid-state brewed vinegar culture
FIG. 2 standard curve of total acid content in fermented grains
Detailed Description
The fermentation stage of Zhenjiang aromatic vinegar is a typical solid-state fermentation process, and the fermentation process of Zhenjiang aromatic vinegar is taken as an example to further explain the invention.
Example 1: extraction of total DNA of microbial community of vinegar culture
1. Collecting samples: collecting vinegar mash samples at different time points in the vinegar fermentation process, and immediately freezing if DNA cannot be extracted in time after the samples are collected.
2. The extraction method of the total DNA of the microorganisms in the vinegar culture comprises the following steps: 5g of vinegar residue was weighed, added with liquid nitrogen in a mortar and sufficiently ground, and transferred to a 50mL centrifuge tube. 13.5mL of DNA extraction buffer and 100. mu.L of lysozyme (50mg/mL) were added to the centrifuge tube and shaken at 37 ℃ for 30min on a 225rpm shaker. 1.5mL of 10% SDS was added to the centrifuge tube, and the mixture was centrifuged at 6000 Xg for 10min at room temperature by gently inverting the mixture every 15-20min in a water bath at 65 ℃ for 2 h. Collecting the supernatant; extracting once with equal volume of chloroform-isoamyl alcohol (24:1, V/V), precipitating L h with 0.6 volume times of isopropanol at room temperature, centrifuging 16000 Xg for 20min, collecting the precipitate, washing the precipitate with precooled 70% ethanol, drying the DNA precipitate, dissolving in 100 μ L of TE, adding RNaseA with the final concentration of 0.5 μ g/mL, digesting in water bath at 37 ℃ for 2h to remove RNA, verifying the effect of DNA extraction by 1.5% agarose gel electrophoresis, and if a band appears at about 10kb, successfully extracting DNA.
example 2: 16S rRNA high-throughput sequencing analysis of microbial community structure of vinegar culture
barcode-PCR amplification
Bacterial 16S rDNA V1-V3Primers used for region PCR amplification can amplify 16S rDNA V1-V3A fragment of about 500bp in length, corresponding to the E.coli 16S rDNA 5 to 534 sites. To facilitate the extraction of the sequence after sequencing, each sample corresponds to a barcode sequence containing 7 bases, i.e., a barcode fragment is added at one end of the primer, and the sequence is as follows:
Forward:5’-XXXXXXX-TGGAGAGTTTGATCCTGGCTCAG-3’
Reverse:5’-XXXXXXX-TACCGCGGCTGCTGGCAC-3’
The barcode-PCR adopted a 25. mu.L system, as follows:
The reaction conditions of barcode-PCR are as follows:
2. High throughput sequencing and analysis
the V1-V3 region of 16S rDNA was amplified by barcode-PCR, the amplified product was further purified, and quantified with PicoGreen after purification. After all amplification products are mixed in equal proportion, high-throughput pyrosequencing is carried out on an FLX Titanium sequencer, and sequencing generates 500bp fragments on average, and the result is shown in figure 1. Analyzing the sequence obtained by sequencing through a mothur software to obtain the species classification information of the microbial community in the vinegar mash and the relative abundance of each species.
Example 3: determination of standard variation curve of total acid content of vinegar grains
and (3) putting 30g of the vinegar mash into a beaker, adding pure water with the volume being 3 times that of the vinegar mash, sealing the cup mouth with a preservative film, stirring and soaking for 3 hours, and filtering by using double-layer filter paper to obtain filtrate for later use. And (3) measuring the total acid content in the vinegar grains according to a NaOH neutralization method in GB/T5009.41-2003 which is an analytical method of edible vinegar sanitary standards.
and tracking and sampling the normal brewing process of randomly selected 30 batches of vinegar, and measuring the total acid content of the fermented grains of vinegar. And fitting the average value of the total acid content of the fermented grains in the 30 batches of vinegar brewing process with the fermentation time to obtain a standard variation curve of the total acid content of the acetic acid (figure 2).
Example 4: application case 1 of microbial vinegar production index
Sampling vinegar residues of the batch A on the 5 th day of normal fermentation, obtaining the relative abundance (Table 1) of microorganisms (Acetobacter and Acinetobacter) which form positive correlation with the total acid of the vinegar residues and microorganisms (Lactobacillus, Xanthomonas, Sphingomonas, Rhizobium, Pantoea, Methylobacterium and Staphyloccus) which form negative correlation with the total acid of the vinegar residues through high-throughput sequencing analysis, and then calculating the acid production index of microbial communities, wherein the calculation process is as follows:
The microbial community acid production index of the 5 th day vinegar residue of the A batch of normal fermentation is [ (0.034+1 × 10)-7)－(0.956+0.0016+0.00046+0.00071+0.00028+4.64×10-5+1×10-7)]-0.1028; substituting the acid production index into the total acid prediction equation of vinegar grain, y-92.09 x2+25.03x +5.729 the predicted value of total acid for 5 days of fermentation batch-92.09 x (-0.1028)2+25.03 × (-0.1028) +5.729 ═ 2.183g/100g dry fermented grains; the total acid of the vinegar grains fermented on the 5 th day of the total acid standard change curve is 2.7g/100g of dry grains, so the acid production of the batch A is relatively slow (2 days slower than the normal fermentation process). According to the prediction result of the total acid of the fermented grains of vinegar on the 5 th day, the total acid content level of the fermented grains of vinegar is gradually improved to be the same as that of the normal fermentation batch by adjusting process parameters such as increasing the fermented grain turning times and the like in the subsequent fermentation process of the batch A, and qualified products can be produced when the fermentation process is finished.
TABLE 1 relative abundance of microorganisms closely related to total acids in the microbial communities of vinegar substrate for Lot A (5 days of fermentation) and Lot B (13 days of fermentation)
Example 5: application case 2 of microbial vinegar production index
Sampling vinegar residues of the 13 th day of fermentation of the batch B, obtaining the relative abundance (Table 1) of microorganisms (Acetobacter and Acinetobacter) which form positive correlation with total acid of the vinegar residues and microorganisms (Lactobacillus, Xanthomonas, Sphingomonas, Rhizobium, Pantoea, Methylobacterium and Staphylococcus) which form negative correlation with the total acid of the vinegar residues through high-throughput sequencing analysis, and then calculating the acid production index of microbial communities, wherein the calculation process is as follows: the acid production index of the vinegar residue of batch B after being normally fermented for 13 days is [ (0.81+0.00026) - (0.066+1 × 10)-7+1×10-7+0.00021+6.51×10-5+0.00013+1×10-7)]0.0827; substituting the index into the prediction equation of total acid of vinegar culture, y is-92.09 x2+25.03x +5.729 the yield of 13 days of fermentation was calculated as-92.09 × (-0.0827)2+25.03 × (-0.0827) +5.729 ═ 7.169g/100g dry fermented grain. The total acid of the fermented grains of vinegar at 5 days of fermentation on the standard change curve of the total acid is 6.7g/100g of dry fermented grains. Therefore, the fermented vinegar of batch B produces acid quickly by microorganisms, and in order to avoid acid volatilization and degradation, the fermentation is stopped for 16 days (2 days before the normal fermentation time). When the finished vinegar finally produced in the batch is analyzed, the total acid content is 6.62g/100mL, wherein the acetic acid content is 3.35g/100mL, and the lactic acid content is 2.31g/100mL, and compared with the quality and the mouthfeel of the finished vinegar of other batches after fermentation is finished for 18 days, the vinegar produced by fermentation in the batch B has no significant difference in quality and mouthfeel. Therefore, the acid production level of microbial fermentation is predicted in the fermentation process through calculation of the acid production index of the microbial community, so that the fermentation is stopped in time, and the fermentation time of 2 days is saved.
Claims (1)
1. A monitoring method for a fermentation process of solid-state brewed vinegar based on acid production index of microbial communities is characterized by comprising the following steps:
(1) Extracting total DNA of the microbial community of the vinegar culture: sampling 100g of a vinegar culture sample from a vinegar factory, fully and uniformly mixing the sample in a sterile plastic package bag, taking 5g of the vinegar culture, and extracting total DNA of a microbial community in the vinegar culture through the steps of liquid nitrogen grinding, lysozyme treatment, chloroform-isoamylol extraction, ethanol washing, RNaseA digestion and the like;
(2) High-throughput sequencing analysis of the microbial community of the vinegar culture: amplifying a 16S rRNA gene V1-V3 region sequence in the total DNA of the microbial community, and quantifying after purifying an amplification product; after all amplification products are mixed in equal proportion, performing high-throughput sequencing on a sequencer, wherein the average length of the sequencing products is 500 bp; obtaining the relative abundance of each type of microorganism in the whole microbial community after sequencing analysis is completed;
(3) calculation of acid production index of microbial community: substituting microorganisms Acetobacter and Acinetobacter which form positive correlation with the total acid of the vinegar culture in the vinegar culture microbial community and microorganisms Lactobacillus, Xanthomonas, Sphingomonas, Rhizobium, Pantoea, Methylobacterium and Staphylococcus which form negative correlation with the total acid of the vinegar culture into a microbial community acid production index calculation equation to obtain a microbial community acid production index, wherein the formula is as follows:
Wherein x is the microbial community acid production index; a (positive correlation) is the abundance of the microorganism which forms a positive correlation with total acid; a (negative correlation) is the abundance of the microorganism forming a negative correlation with total acid; m is the number of microbial species forming a positive correlation with total acid; n is the number of microbial species having a negative correlation with total acid formation;
(4) Predicting the total acid content of the vinegar grains: substituting the acid production index of the microbial community obtained by calculation in the step (3) into a total acid content prediction equation of the vinegar substrate, so as to predict the total acid content of the vinegar substrate according to the relative abundance of microbes; the prediction equation is as follows:
y＝-92.09x2+25.03x+5.729
Wherein y is a predicted value of the total acid content of the vinegar grains, calculated according to g/100g of dry grains, x is an acid production index of a microbial community, and the fitting coefficient of the equation is 0.895;
(5) Judging the fermentation process of the solid-state brewed vinegar: comparing the predicted value of the total acid of the fermented grains with the standard curve of the total acid change of the fermented grains, so as to judge and predict the actual progress of the batch of solid-state fermented vinegar, guide the adjustment of vinegar fermentation parameters and predict and judge the fermentation end time; wherein the standard variation curve of the total acid of the fermented grains is obtained by fitting the average value of the total acid content of the fermented grains in the solid state brewing of more than 30 batches of normal vinegar; the total acid content of the vinegar grains is measured according to a NaOH neutralization method in GB/T5009.41.
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