| Currently, low research level and lacking of systematic theoretical system of Sichuan dongcai have become main factors restricting its industrialization development. Therefore, it‘s urgently to do systematic theoretical research on Sichuan dongcai. In this study, microbial community structures in Sichuan dongcai at different salting times were analyzed to explore dynamic changes law using metagenome or traditional pure culture methods; and species and relative abundances of dominant microorganisms in each salting period were identified. Nitrite, crude protein, amino acid contents, color and micro-morphology changes in Sichuan dongcai at different salting times were analyzed, and a more comprehensive description of quality and appearance variation laws during the entire salting process was given. Volatile compounds and flavor profile of Sichuan dongcai at different salting times were investigated using solid phase microextraction combined with gas chromatography–mass spectrometry(SPME–GC–MS) and an electronic nose(E-nose); and samples from different salting times were successfully discriminated by principal component analysis(PCA). The relationships between microorganisms and nitrite, amino acids, color as well as flavor of Sichuan dongcai during salting process were analyzed based on metagenome functional annotation results. The main contents and results of this study are as follows: 1. Microbial community structure analysis of Sichuan dongcai(1) To fully grasp dynamic changes law of microbial community structure during salting process of Sichuan dongcai, metagenome or traditional pure culture methods were used for analyzing; both species number and relative abundances of identified bacteria were much greater than that of fungi. It showed that the main microorganisms presenting in Sichuan dongcai were bacteria rather than fungi, and the total amount of microorganisms decreased with salting time.(2) A total of 85 bacteria and 5 fungi were identified in four dongcai samples. In samples of 0#~3#, bacterial species(including subspecies) numbers were 55, 36, 31 and 4, respectively; fungi species numbers were 1, 2, 2 and 1, respectively.(3) There was 1 microorganism(Lactobacillus brevis) identified in all four samples. There were 10 bacteria simultaneously identified in samples of 0#~2#, including 3 Lactobacillus, 1 Lactococcus, 4 Leuconostoc, 1 Psychrobacter and 1 Weissella. There were 22 microorganisms simultaneously identified in two samples, including 19 bacteria and 3 fungi.(4) There were 24 microorganisms simultaneously presented in 1# and 2# samples, and accounted for 63% and 71% of total microbial species numbers, respectively. Moreover, 8 of the top ten microorganisms in relative abundance were the same in samples of 1# and 2#. It indicated that microbial community structures in samples 1# and 2# were similar, and microbial species in Sichuan dongcai had small changes during 1~2 years salting.(5) Pseudomonas and Arthrobacter were dominant microorganisms in no-salting dongcai(0#), and their relative abundances were 21.98% and 6.74%, respectively. In medium-saltingperiod(1# and 2#), dominant microorganisms was Lactobacillus, whose relative abundance reached 87.88% and 68.86% in the two samples, respectively. Relative abundanceof L. brevis was highest, and reached 84.19% and 65.97% in the two samples, respectively. Microorganism species number was small in post-salting period(3#), and Bacillus was the possible dominant microorganism in the period. 2. Quality and appearance analysis of Sichuan dongcai(1) To understand the safety of Sichuan dongcai, nitrite that is usually consideredas a harmful substance in pickled food was selected as a monitoring indicator and its content changes law during salting process was studied. It is found that nitrite content was on the decline during salting process. Nitrite content reduced by 70% at salting time of 6 months compared with no-salting one; the content was only 1 mg/kg dry matter~2 mg/kg dry matter which was far below relevant national standard limit after 30 months salting.(2) To understand the nutritional value changes of Sichuan dongcai, change laws of crude protein and amino acid contents were studied. It showed that crude protein, total and essential amino acid contents were on the decline during salting process. Crude protein content reached 30 g/100 g dry matter in no-salting dongcai, and reduced by 78% to 6.6 g/100 g dry matter at salting time of 36 months. Essential amino acids proportion rose from 23% to 33%~36% after 6 months salting, and maintained at a steady level. Both glutamic acid and proline(non-essential amino acid) contents were higher than total essential amino acids content in no-salting dongai, but their contents reduced by 48% and 89% during the first 6 months salting, respectively. It directly resulted in the reduction of non-essential amino acid proportion.(3) To understand appearance and morphology changes of Sichuan dongcai, color and micro-morphology changes during salting process were studied. It was found that the color was different between three parts which were concave side of stem, convex side of stemand leaves at beginning, but color of the whole dongcai was uniform after long time salting. The color was changing from light to dark, green to red and yellow to blue, which was consistent with observed changes by naked eyes.Persons not sensitive to color change(can distinguish color change when ΔEab*>5.0) would think that there was no difference in color between three parts of dingcai at medium- and post-salting periods(24~39 months); even persons sensitive to color change(can distinguish color change when ΔEab*>3.0) would think that there was no difference in color between three parts at salting time of 39 months. Structure of sample tissue was gradually blurred and damaged observed by micro-morphology. 3. Flavor analysis of Sichuan dongcai(1) To find out characteristic compounds related to unique flavor of Sichuan dongcai, volatile compounds during salting process were analyzed using SPME–GC–MS. A total of 70 volatile compounds were identified in four samples from different salting times. In dongcai samples of 0#~3#, 21, 8, 32 and 38 compounds were detected, respectively, and their contents were 379 μg/g dry matter、44 μg/g dry matter, 968 μg/g dry matter and 325 μg/g dry matter, respectively.(2) There are 21 volatile compoundsdetected in at least two periods. These compounds, which may play an important role in flavor, represented 77.3%, 96.5%, 89.9% and 78.9% of the total volatile compounds in the four periods, respectively. Allyl isothiocyanate, phenylethyl alcohol and benzaldehyde were identified in all four periods; limonene and nonanal were identified in three periods; while 16 volatile compounds were identified in two periods, and they were toluene,(Z)-β-ocimene, α-cubebene, caryophyllene, β-gurjunene, δ-cadinene, phenol, eugenol, hexanoic acid, 4-hexadecyl ester, estragole and anethole, hexanal,(E,E)-2,4-Heptadienal, Benzeneacetaldehyde, acetophenone and β-Ionone.(3) To find out characteristic compounds related to flavor of Sichuan dongcai in different salted periods, the 70 detected volatile compounds and 21 common compounds were used for PCA, respectively. PCA results indicated that both the 70 and the 21 compounds could clearly discriminate the four samples. Flavor characteristics of sample 0# mainly depended on sulphur cyanogen compounds, alcohols and aldehydes; flavor characteristics of sample 1# mainly depended on toluene and hexanoic acid, 4-hexadecyl ester; flavor characteristics of sample 2# mainly depended on hydrocarbon especially terpene; flavor characteristics of sample 3# mainly depended on aldehydes and heterocyclic compounds.(4) To discriminate Sichuan dongcai samples at different salting times, flavor profile of samples in different periods was analyzed using E-nose. Results showed that flavor differences could be used to determine salting degree of Sichuan dongcai. And the chamber 1 sensors especially LY2/gCTL, LY2/GH, LY2/AA and LY2/G were used for sample discrimination. 4. Microbial metagenome analysis(1) For further analysis of metagenome annotation information of microorganisms in Sichuan dongcai, annotation results statistics based on KEGG and eggNOG databases were made, respectively. Annotated metabolic pathways on KEGG were 256, 197 and 215 in samples of 0#, 1# and 2#, respectively. Among these pathways, there were 178 ones were simultaneously annotated in all three samples. The main microorganisms annotated on KEGG pathways were lactobacillus and pseudomonas. There were 23, 20 and 21 COGs functional classifications based on eggNOG in 0#, 1# and 2#, respectively. Among these functional classifications, there were 20 ones were the same in all three samples.(2) To study the relationship between microorganisms and nitrite degradation, nitrite degradation pathways as well as annotation of genes were analyzed. Lactobacillus reuteri, Streptococcus agalactiae, Alcanivorax borkumensis and Halomonas maurawere related to nitrite metabolism in Sichuan dongcai. Nitrite degradation pathways can be summarized as follows: in samples of 1# and 2#, extracellular nitrite is transferred into cell, then transforms into ammonia under the action of reductase; or transforms into glutamine or glutamate by glutamine synthetase or glutamate synthase, respectively; next participates in glutamate metabolism pathway; further it transforms into succinate, 1-pyrroline-5-carboxylic acid and phosphorus amine compounds under the action of different enzymes; these substances participate in other metabolism pathways at last. Besides the above pathways, nitrite can be degraded to nitric oxide and nitrous oxide by a series of reductases in sample 0#.(3)To study the relationship between microorganisms and amino acids metabolism, metabolism pathways as well as annotation of genes were analyzed. Microbial species related to amino acids metabolism is divers. Besides Lactobacillus and pseudomonas, there were other Bacillus and Coccus, etc. These microorganisms mainly took part in amino acids degradation activities, while contributed less to amino acids synthesis. Conversions between amino acids may be one of the reasons for ratio changes in essential amino acids and non-essential amino acids.(4) To study the relationship between microorganisms and color, potential metabolism pathways related to color changes as well as annotation of genes were analyzed. Only Pseudomonas mendocina was annotated on KO00860 pathway in sample 0#. On this pathway, one more oxygen-dependent protoporphyrinogen oxidase(EC: 1.3.3.4) was annotated in sample 2# rather than 0# and 1#. This enzyme canoxidize colorless protoporphyrinogen IX to protoporphyrin IX, which may be one of the reasons that sample 2# looks darker than 1#.(5) To study the relationship between microorganisms and flavor, potential metabolism pathways related to flavor compounds as well as annotation of genes were analyzed. On the assumption that the detected volatile compounds were associated with flavor of Sichuan dongcai, annotated microorganisms related to flavor were Lactobacillus, Pseudomonas, Monoucleosis and Coccus, etc. Terpene biosynthesis has two pathways including MP and MEP/DOXP on KO00900. Metabolites of microorganisms in Sichuan dongcai participated in KO00902, KO00909 and KO00130 by MEP/DOXP and MP in samples of 0# and 1#, respectively; and participated in KO00902 and KO00909 by MP in samples 2#. |
PDF Full Text Request