CN105349690B - A method for monitoring the fermentation process of solid-state brewing vinegar based on microbial community acid production index - Google Patents

Abstract

The invention belongs to the field of biological fermentation, and in particular relates to a method for monitoring the fermentation process of solid-state brewing vinegar based on the acid production index of microbial communities. In order to overcome the deficiencies of the traditional monitoring method for the fermentation process of vinegar brewing, the present invention uses high-throughput sequencing technology to measure the microbial community structure of vinegar fermented grains in the solid-state fermentation process of vinegar, and then uses the relative abundance of microorganisms related to the formation of total acid The acid production index of the microbial community was calculated by using the numerical value, and then substituted into the total acid prediction equation of the vinegar grains to calculate the predicted value of the total acid content of the vinegar grains sample. After comparing with the standard change curve of the total acidity of the vinegar grains, it can be known that the batch fermentation process is biased. Whether it is fast or slow, according to the results, the fermentation process parameters can be adjusted in time, and the fermentation end time can be judged and predicted in time.

Description

A fermentation process monitoring of solid-state brewing vinegar based on microbial community acid production index method

technical field

The invention belongs to the field of biological fermentation, and in particular relates to a method for monitoring the fermentation process of solid-state brewing vinegar based on the acid production index of microbial communities.

Background technique

The production of vinegar in my country has a long history. The brewing process is a multi-strain mixed fermentation process, through the metabolism of various microorganisms to produce vinegar with full flavor and soft sour taste. There are two main fermentation stages in the production of vinegar: alcoholic fermentation and acetic fermentation. In the stage of alcohol fermentation, starchy raw materials such as glutinous rice and sorghum go through a series of metabolisms of mold and yeast, and finally produce alcohol; in the stage of acetic acid fermentation, the key step to determine the flavor and quality of vinegar is that many microorganisms participate in this process Fermentation converts ethanol into acids and many other flavor substances, so the microbial flora is the guarantee of the flavor quality of solid-state brewed vinegar products.

At present, the vinegar fermentation process control of many vinegar manufacturers in my country is mainly based on traditional operating experience, mainly according to the fixed fermentation method and fermentation time. The determination of the end point of the acetic acid fermentation process depends on physical and chemical indicators such as the content of metabolites such as total acid and non-volatile acid, so the controllability of the entire solid-state acetic acid fermentation process is not high. The total acid, non-volatile acid, and volatile flavor substances in vinegar grains are all the result of the metabolic activities of vinegar-making microorganisms. Therefore, tracking the solid-state fermentation process of vinegar through total acid and other metabolite indicators often lags behind. Low total acid and non-volatile acid content make it difficult to adjust the process in time during the fermentation process, resulting in batch-to-batch differences in product quality, especially large differences in product flavor. In the process, only the content of total acid and non-volatile acid is monitored, and the process parameters cannot be adjusted in time according to the changes of microorganisms in the fermentation process. In addition, total acid, non-volatile acid, and flavor substances are all metabolites of microorganisms. There is a lag in the change process compared with it.

With the rapid development of high-throughput sequencing technology for microbial communities, people can quickly study the type, quantity and function of microorganisms in a specific environment from the genetic level, which helps people to understand the information of complex microbial environments to a large extent. Many researchers at home and abroad have applied high-throughput sequencing technology to the microecological research of prokaryotes and eukaryotes in feces, soil, seabed sludge, sewage and other systems, and it has also been widely used in the field of traditional fermented food. These include studies on the microbial composition and distribution of Mexican fermented corn balls, Japanese black vinegar, Korean kimchi, cheese Italian sausage, etc., and have achieved remarkable results, providing a theoretical basis for us to better understand the mechanism of traditional fermentation at home and abroad At the same time, it also provides valuable theoretical data support for the reform and progress of the traditional fermentation industry.

The invention provides a method for monitoring the fermentation process of solid-state brewing vinegar based on the acid production index of microbial communities. This method uses high-throughput sequencing technology to measure the microbial community structure of vinegar fermented grains in the middle of the solid-state fermentation process, and then uses the relative abundance value of microorganisms related to the formation of total acid to calculate the microbial community acid production index, and substitute it into the forecast of total acid in vinegar grains The equation calculates the predicted value of the total acid content of the vinegar grain sample, and by comparing it with the standard change curve of the vinegar grain total acid, it can be known whether the fermentation process of the batch is fast or slow, and the fermentation process parameters can be adjusted in time according to the results, and Timely judge and predict the end time of fermentation. .

Contents of the invention

The purpose of the present invention is to solve the above-mentioned deficiencies in the controllability and unstable batch quality of the existing vinegar solid-state brewing process, and to provide a method for monitoring the solid-state fermentation of vinegar based on the mechanism of microbial acid production and the method of microbial community acid production index The accuracy of this method is relatively high, which has laid a theoretical foundation for the transformation of my country's solid-state vinegar brewing industry from "experience-based" control to "scientific and technological-based".

The present invention is realized through the following scheme: first, sample the vinegar unstrained spirits sample in the solid-state fermentation vinegar fermentation process, and extract the total DNA of the microbial community in the vinegar unstrained grains sample, and use high-throughput sequencing technology to mark the genetic development of the sample genome The V1-V3 regions of 16S rDNA were subjected to high-throughput sequencing, and the relative abundance values of microorganisms in the microbial community of vinegar fermented grains that were closely related to the formation of total acid in vinegar grains were substituted into the calculation equation for the acid production index of the microbial community, and the calculated production The acid index was substituted into the prediction equation of the total acid content of the vinegar grains to obtain the predicted value of the total acid content of the vinegar grains. The predicted value of the total acidity of the vinegar grains was compared with the standard curve of the change of the total acidity of the vinegar grains, so that the actual progress of the batch of solid-state fermentation vinegar could be judged and predicted, and the adjustment of the vinegar fermentation parameters could be guided. The method monitors the vinegar brewing process by monitoring the abundance of microorganisms related to acid production in vinegar fermented grains. The prediction accuracy of acid production is high, and it has a good monitoring effect on the quality of vinegar. The specific implementation route is as follows :

(1) Extraction of the total DNA of the microbial community of fermented vinegar grains: 100 g of fermented grains of vinegar was sampled from a vinegar brewery, mixed thoroughly in a sterile plastic bag, and 5 g of fermented grains of vinegar were taken, ground with liquid nitrogen, treated with lysozyme, and treated with chloroform-isoamyl Alcohol extraction, ethanol washing, RNaseA digestion and other steps were used to extract the total DNA of microbial communities in vinegar fermented grains.

(2) High-throughput sequencing analysis of the microbial community of vinegar fermented grains: the sequences of the V1-V3 regions of the 16S rRNA gene in the total DNA of the microbial community were amplified, and the amplified products were purified and quantified. All amplified products were mixed in equal proportions and then subjected to high-throughput sequencing on a sequencer. The average length of the sequenced products was 500bp. After the sequencing analysis is completed, the relative abundance of various microorganisms in the entire microbial community is obtained.

(3) Calculation of the acid production index of the microbial community: the microorganisms (Acetobacter, Acinetobacter) that have a positive correlation with the formation of total acid in vinegar grains and the microorganisms (Lactobacillus, Xanthomonas) that have a negative correlation with the formation of total acid in vinegar grains (Lactobacillus, Xanthomonas) in the vinegar grains microbial community , Sphingomonas, Rhizobium, Pantoea, Methylobacterium, Staphylococcus) were substituted into the calculation equation of microbial acid production index to obtain the microbial community acid production index.

(4) Prediction of the total acid content of the vinegar grains: Substitute the acid production index of the microbial community calculated in the above step (3) into the prediction equation of the total acid content of the vinegar grains, so that the total acid content of the vinegar grains can be predicted according to the relative abundance of microorganisms .

(5) Judgment of the fermentation process of solid-state fermented vinegar: compare the predicted value of the total acidity of the vinegar grains obtained in the above step (4) with the standard curve of the change of the total acidity of the vinegar grains, so that the quality of the batch of solid-state fermented vinegar can be judged and predicted. Actual progress, and guide the adjustment of vinegar fermentation parameters.

The invention provides a method based on microbial community acid production index to monitor the fermentation process of vinegar solid-state fermentation. By measuring the relative abundance of microbial communities and calculating the acid production index to judge whether the fermentation process is slow, normal or fast, it can predict the trend of fermentation, help to adjust process parameters in time, and determine the end time of fermentation in advance.

Description of drawings:

Figure 1 Sequence length distribution of DNA pyrosequencing of microbial community of vinegar fermented grains for solid-state brewing

Figure 2 Standard change curve of total acid content in vinegar fermented grains

Detailed ways

The acetic acid fermentation stage of Zhenjiang balsamic vinegar is a typical solid-state fermentation process, and the present invention will be further described by taking the acetic acid fermentation process of Zhenjiang balsamic vinegar as an example.

Embodiment 1: Extraction of total DNA of vinegar fermented grain microbial community

1. Sample collection: collect vinegar unstrained spirits samples at different time points during the vinegar fermentation process. If the DNA cannot be extracted in time after sample collection, it should be frozen immediately.

2. Extraction method of microbial total DNA in vinegar fermented grains: Weigh 5g of vinegar fermented grains, add liquid nitrogen in a mortar and grind thoroughly, transfer to a 50mL centrifuge tube. Add 13.5 mL of DNA extraction buffer and 100 μL of lysozyme (50 mg/mL) to the centrifuge tube, and shake at 225 rpm for 30 min at 37° C. on a shaker. Add 1.5mL 10% SDS to the centrifuge tube, bathe in water at 65°C for 2h, invert gently every 15-20min, and centrifuge at 6000×g for 10min at room temperature. Collect the supernatant; extract once with an equal volume of chloroform-isoamyl alcohol (24:1, V/V), precipitate with 0.6 times the volume of isopropanol at room temperature for 1 h, centrifuge at 16,000×g for 20 min, collect the precipitate, and use a pre-cooled Wash the precipitate with 70% ethanol, dissolve the DNA precipitate in 100 μL TE after drying, add RNaseA at a final concentration of 0.5 μg/mL, and digest it in a water bath at 37°C for 2 hours to remove RNA, and verify the DNA by 1.5% agarose gel electrophoresis The effect of extraction, if a band appears around 10kb, the DNA extraction is successful.

Example 2: 16S rRNA high-throughput sequencing analysis of microbial community structure of vinegar fermented grains

1. Barcode-PCR amplification

The primers used for the PCR amplification of the bacterial 16S rDNA V ₁ -V ₃ region can amplify a fragment of about 500 bp in the 16S rDNA V ₁ -V ₃ region, corresponding to sites 5 to 534 of E.coli 16S rDNA. In order to facilitate the sequence extraction after sequencing, each sample corresponds to a barcode sequence containing 7 bases, that is, a barcode fragment is added to one end of the primer, and the sequence is as follows:

Forward: 5’-XXXXXXX-TGGAGAGTTTGATCCTGGCTCAG-3’

Reverse: 5’-XXXXXXX-TACCGCGGCTGCTGGCAC-3’

The barcode-PCR uses a 25 μL 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, and the amplified product was further purified, and quantified by Picogreen after purification. All amplified products were mixed in equal proportions and then subjected to high-throughput pyrosequencing on the FLX Titanium sequencer. Sequencing produced an average of 500 bp fragments. The results are shown in Figure 1. The sequence obtained by sequencing was analyzed by mothur software, and the species and classification information of the microbial community in the vinegar fermented grains and the relative abundance of each species were obtained.

Embodiment 3: the mensuration of the standard change curve of total acid content of vinegar fermented grains

Take 30g of vinegar grains in a beaker, add 3 times the volume of pure water, seal the mouth of the cup with plastic wrap, stir and soak for 3h, filter with double-layer filter paper, and the filtrate is set aside. According to the NaOH neutralization method in "Vinegar Hygienic Standard Analysis Method" GB/T 5009.41-2003, the total acid content in vinegar fermented grains was determined.

30 randomly selected batches of vinegar during the normal brewing process were tracked and sampled, and the total acid content of vinegar fermented grains was determined. The standard change curve of the total acid content of acetic acid was obtained by fitting the average value of the total acid content of the vinegar fermented grains and the fermentation time in the 30 batches of vinegar brewing process (Fig. 2).

Embodiment 4: the application case 1 of microbial vinegar production index

Sampling of the vinegar grains on the 5th day of normal fermentation in batch A, through high-throughput sequencing analysis, it was found that the microorganisms (Acetobacter, Acinetobacter) that were positively correlated with the formation of total acid in vinegar grains and negatively correlated with the formation of total acid in vinegar grains The relative abundance of microorganisms (Lactobacillus, Xanthomonas, Sphingomonas, Rhizobium, Pantoea, Methylobacterium, Staphylococcus) (Table 1), and then calculate the microbial community acid production index, the calculation process is as follows:

Microbial community acid production index of vinegar fermented grains in batch A of normal fermentation on day 5 = [(0.034+1×10 ^-7 )－(0.956+0.0016+0.00046+0.00071+0.00028+4.64×10 ^-5 +1×10 ^-7 )]/9=-0.1028; Substituting the acid production index into the total acid prediction equation of vinegar fermented grains y=-92.09x ² +25.03x+5.729 can calculate the total acid prediction value=-92.09×( -0.1028) ² +25.03×(-0.1028)+5.729＝2.183g/100g dry unstrained spirits; and the total acid of vinegar unstrained spirits fermented on the fifth day on the total acid standard curve is 2.7g/100g dry unstrained spirits, so batch A Acid production is relatively slow (2 days slower than the normal fermentation process). According to the prediction results of the total acidity of the vinegar grains on the fifth day, in the subsequent fermentation process of batch A, by adjusting the process parameters such as increasing the number of turning over grains, the level of the total acid content of the vinegar grains was gradually increased to be the same as that of the normal fermentation batch. At the end of the process, qualified products can be produced.

Table 1 The relative abundance of microorganisms closely related to total acid in the microbial community of vinegar fermented grains in batch A (fermentation 5 days) and batch B (fermentation 13 days)

Embodiment 5: Application Case 2 of Microbial Vinegar Production Index

The vinegar fermented grains on the 13th day of B batch fermentation were sampled and analyzed by high-throughput sequencing, and the microorganisms (Acetobacter, Acinetobacter) that were positively correlated with the formation of total acid in vinegar grains and the microorganisms that were negatively correlated with the formation of total acid in vinegar grains were obtained. The relative abundance of microorganisms (Lactobacillus, Xanthomonas, Sphingomonas, Rhizobium, Pantoea, Methylobacterium, Staphylococcus) (Table 1), and then calculate the microbial community acid production index, the calculation process is as follows: B batch of acid production index of vinegar fermented grains fermented normally for 13 days ＝[(0.81+0.00026)－(0.066+1×10 ^-7 +1×10 ^-7 +0.00021+6.51×10 ^-5 +0.00013+1×10 ^-7 )]/9＝0.0827; substitute this index into vinegar The total acid prediction equation of fermented grains y=-92.09x ² +25.03x+5.729 can be used to calculate the acid production of this batch of fermentation for 13 days=-92.09×(-0.0827) ² +25.03×(-0.0827)+5.729=7.169g /100g dry unstrained spirits. On the total acid standard change curve, the total acid of the vinegar fermented grains fermented on the 5th day was 6.7g/100g dry fermented grains. Therefore, the vinegar fermented grain microorganisms in batch B produced acid quickly. In order to avoid acid volatilization and degradation, we stopped the fermentation on 16 days (2 days earlier than the normal fermentation time). The final output of this batch of vinegar was analyzed. The total acid content was 6.62g/100mL, of which the acetic acid content was 3.35g/100mL and the lactic acid content was 2.31g/100mL. Compared with the quality and taste, the vinegar produced by B batch fermentation has no significant difference in quality and taste. Therefore, through the calculation of the microbial community acid production index, the level of microbial fermentation acid production was predicted during the fermentation process, so that the fermentation was terminated in time, saving 2 days of fermentation time.

Claims (1)

1. a monitoring method based on the fermentation process of solid-state brewing vinegar based on microbial community acid production index, characterized in that the monitoring method is carried out according to the following steps: (1) Extraction of the total DNA of the microbial community of fermented vinegar grains: 100 g of fermented grains of vinegar was sampled from a vinegar brewery, mixed thoroughly in a sterile plastic bag, and 5 g of fermented grains of vinegar were taken, ground with liquid nitrogen, treated with lysozyme, and treated with chloroform-isoamyl Alcohol extraction, ethanol washing, RNaseA digestion and other steps to extract the total DNA of the microbial community in the vinegar fermented grains; (2) High-throughput sequencing analysis of the microbial community of vinegar fermented grains: the sequence of the V1-V3 region of the 16S rRNA gene in the total DNA of the microbial community was amplified, and the amplified products were purified and quantified; all amplified products were mixed in equal proportions and sequenced High-throughput sequencing is performed on the instrument, and the average length of the sequencing product is 500bp; after the sequencing analysis is completed, the relative abundance of various microorganisms in the entire microbial community is obtained; (3) Calculation of the acid production index of the microbial community: Acetobacter and Acinetobacter, which are positively correlated with the formation of total acid in vinegar grains, and Lactobacillus, Xanthomonas, and Sphingomonas, which are negatively correlated with the formation of total acid in vinegar grains in the vinegar grains microbial community , Rhizobium, Pantoea, Methylobacterium, and Staphylococcus were substituted into the calculation equation of the acid production index of the microbial community to obtain the acid production index of the microbial community, the formula is as follows: Among them, x is the acid production index of microbial community; A (positive correlation) is the microbial abundance that has a positive correlation with the total acid formation; A (negative correlation) is the microbial abundance that has a negative correlation with the total acid formation; m is the microbial abundance that has a negative correlation with the total acid formation The number of microbial species with positive correlation with total acid formation; n is the number of microbial species with negative correlation with total acid formation; (4) Prediction of the total acid content of the vinegar grains: Substitute the acid production index of the microbial community calculated in the above step (3) into the prediction equation of the total acid content of the vinegar grains, so that the total acid content of the vinegar grains can be predicted according to the relative abundance of microorganisms ; The prediction equation is as follows: y=-92.09x ² +25.03x+5.729 Wherein y is the predicted value of the total acid content of vinegar fermented grains, calculated by g/100g dry fermented grains, x is the acid production index of microbial community, and the fitting coefficient of this equation is 0.895; (5) Judgment of the fermentation process of solid-state fermented vinegar: compare the predicted value of the total acidity of the vinegar grains obtained in the above step (4) with the standard curve of the change of the total acidity of the vinegar grains, so that the quality of the batch of solid-state fermented vinegar can be judged and predicted. Actual progress, and guide the adjustment of vinegar fermentation parameters, predict and determine the end time of fermentation; the standard change curve of the total acid content of vinegar grains is based on the average value of the total acid content of vinegar grains in more than 30 batches of normal vinegar solid-state brewing Fit the obtained standard variation curve; the total acid content of the vinegar fermented grains is determined according to the NaOH neutralization method in GB/T 5009.41.