**1. Introduction**

Grapes are fresh and juicy, bright in color, sweet and sour and rich in nutritional value [1]. However, because they are harvested in the high-temperature season, the fruit stem is a typical respiratory climacteric type [2], the physiological metabolism is vigorous after harvest, and the fruit tissue is crisp and tender. During storage, it is very prone to water loss, stem withering, browning and easily infected by bacteria and rot, resulting in a short storage cycle, which seriously affects the appearance quality and commodity value of fresh grapes, causing huge economic losses and food waste. At present, the preservation measures of grapes at home and abroad mainly use sulfur dioxide (SO2) preservatives, coating preservation, lowtemperature refrigeration and other technologies to prolong the storage period of grapes [3]. Although SO2 has a good effect on maintaining the quality of grapes, it is easy to cause certain bleaching damage to the fruit, affect the original flavor, and the SO2 remaining in the fruit will be harmful to human health [4]. Packaged food preservation is one of the effective

**Citation:** Hu, B.; Lin, L.; Fang, Y.; Zhou, M.; Zhou, X. Application of Chitosan-Lignosulfonate Composite Coating Film in Grape Preservation and Study on the Difference in Metabolites in Fruit Wine. *Coatings* **2022**, *12*, 494. https://doi.org/ 10.3390/coatings12040494

Academic Editor: Elena Poverenov

Received: 28 February 2022 Accepted: 30 March 2022 Published: 7 April 2022

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**Copyright:** © 2022 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/).

solutions. However, the packaging material commonly used in the preservation process is plastic; for example, PE, PS, or PET [5]. Chitosan is a kind of natural edible macromolecular polysaccharide that is non-toxic, has no smell, with good film forming characteristics [6,7]. After coating treatment, a colorless and transparent biological film is formed on the grape surface, which can prevent water loss and reduce the rate of weight loss in the fruit. It can also effectively prevent microbial invasion, reduce the respiratory intensity of the fruit [8–10] to a certain extent, and reduce the decay of the fruit, thus prolonging its storage time [11]. However, the coating effect of single chitosan is not stable [12], the drying time is long, the water permeability is high, and the coating toughness is poor. Sodium lignosulfonate is an anionic polymer surfactant extracted from papermaking waste liquor [13]. Because of its similar structure to lignin, it is often used as a bio-based polymer material [14]. LS has a good function but cannot form a film by itself. We tried adding LS to CH to make films suitable for fruit preservation, because the use of bio-based materials for packaging can not only be used as cheap packaging for fresh fruits and vegetables, but also reduce the pollution of plastic packaging to the environment. It achieves the requirements of highly efficient anticorrosion, low carbon and environmental protection.

Wine is fresh grapes or grape juice as raw materials, through all or part of the fermentation brewing, containing a certain degree of alcohol fermented wine [15]. It is not only a nutritious beverage, but moderate drinking can prevent various chronic diseases and enhance human health [16–18]. Fresh grapes are the key raw materials for wine brewing, and the style of wine is closely related to the variety of wine grapes, the climate of the place of origin, soil conditions and the distinctive brewing processes, making the wines produced in different producing areas have different flavors [19]. The eastern foot of Helan Mountain in Ningxia is located in the "golden" area of grape planting at 30 to 40 latitudes from north to south. The superior geographical location, unique landform features, suitable soil and climate conditions mean that wine grapes in the eastern foot of Helan Mountain in Ningxia fully possess excellent brewing characteristics.

With the rapid development of the world economy and the improvement of people's quality of life, the wine industry has developed rapidly and has become an increasingly popular product for consumers [20], but the consumer's ability to distinguish is limited. Usually, physical and chemical indexes and sensory evaluation are used to identify the quality of wine grapes and wine, but it is difficult to reflect various metabolites in wine grapes and wine that affect their quality and are beneficial to human health through these indicators [21]. In studies on wine metabolites, a "metabolic fingerprint" is generally provided based on 1H-NMR technology [22,23]. Gregory et al. [24] used NMR to analyze and study metabolites in French red wine in the region of Bordeaux, so as to better distinguish red wine of Bordeaux from other red wines produced in French wine regions. In chemometrics, pattern recognition is the main method used to solve the attribution problem and marker search in complex systems, among which PLS-DA is the most important pattern recognition method applied in metabonomics. It is widely used in plant, drug metabolomics and food source determination and classification research [25]. Godelmann R. et al. [26] used nuclear magnetic resonance technology combined with multivariate statistics and principal component analysis to analyze and study the target compounds and non-target compounds of wine metabolites, and the results showed that the accuracy of variety identification reached 95%, the accuracy of age identification reached 97% and 96%, respectively, and the accuracy of origin identification reached 89%. All these results showed that 1H-NMR combined with multivariate analysis was an extraordinary effective method to identify different wine varieties and region.

This paper investigated the preparation method of separate 2% chitosan film (CH), chitosan—1% sodium lignosulfonate film (1% CH/LS) and chitosan—2% sodium lignosulfonate film (2% CH/LS), and the film forming properties of the three films were studied. The preservation effect of 2% CH/LS film on grape berry was then studied further and compared with PE film (Control) and Chitosan film (CH). The differences in metabolites in Cabernet Sauvignon, Chardonnay and Italian Riesling wines at the eastern foot of Helan

Mountain in Ningxia were also studied to determine the biomarkers that contribute to the differences, so as to provide the cornerstone for wine quality control, variety identification and protection, Additionally, we also provide a scientific theoretical basis for consumers to choose high quality wine.

### **2. Materials and Methods**

### *2.1. Reagents and Equipment*

Chitosan(C6H11NO4, Average molecular weight (MW): 150 kDa; Degree of deacetylation ≥ 90%) were purchased from Shanghai Lanji Biological Technology Co., Ltd. (Shanghai, China); Sodium-Lignosulfonate (Content ≥ 98%) were purchased from Hefei BOSF Biotechnology Co., Ltd. (Hefei, China); Ascorbic acid (Vc) was obtained from Sinopharm Group Chemical Reagent Co., Ltd. (Shanghai, China); Oxalic acid, Sodium oxalate were produced by Su Yi Chemical Reagent Co., Ltd., Shanghai, China; DSS was produced by Qingdao Tenglong Microwave Technology Co., Ltd. (Qingdao, China); Heavy water (D2O deuterium degree > 99.9%) is from Tenglong Weibo Technology Co., Ltd., Qingdao, China.

SNL315SV-230 Freeze dryer was produced by Termo Co., Ltd., Waltham, MA, USA; AVANCE 600 Nuclear magnetic resonance spectrometer was supplied by Bruker Co., Ltd., Karlsruhe, German; TG16A-WS Desktop high-speed centrifuge was produced by Lu Xiangyi Centrifuge Instrument Co., Ltd., Shanghai, China; BS-224 Electronic balance was produced by Eppendorf Co., Ltd., Hamburg, German; ULT178-6-V49 Ultra low temperature freezer was supplied by Revco Co., Ltd., Waltham, MA, USA; RE52-4 Rotary evaporator was produced by Huxi Analysis Instrument Co., Ltd., Shanghai, China; XW-80A Swirl mixer was supplied by Huxi Analysis Instrument Co., Ltd., Shanghai, China.

### *2.2. Methods*

### 2.2.1. Preparation Method CH/LS Bio-Composite Film

LS powder (1 g) was dissolved in 50 mL of distilled water at 5000 rpm for 15 min. CH powder (1 g) was dissolved in 50 mL of distilled water at 5000 rpm for 15 min at 25 ◦C. Then, VC (1 g) and chitosan was added at 5000 rpm for 15 min by stirring continuously. Finally, the CH/LS film-forming solutions were degassed to remove air bubbles [27]. The 20 mL CH/LS film-forming solution was cast on a 15 cm in diameter flat glass Petri dish (B-SLPYM90, BKMAM Biological Co., Ltd., Changsha, China) and dried in an oven at 35 ◦C for 24 h until the surface of CH/LS film remained certain firm and viscous. Finally, the CH/LS film was stored in a small desiccator at 50% relative humidity for 20 min at 25 ◦C. Peel the CH/LS film from the Petri dish for analysis. The prepared films were, respectively added with 0% LS + 2% CH, 1% LS + 2% CH, 2% LS + 2% CH, so they were named as CH/LS, 1% CH/LS, 2% CH/LS, respectively [28].

### 2.2.2. Determination Method of CH/LS Film on the Preservation Effect of Grape Berry

Seasonal fresh grapes (Cabernet Sauvignon variety, from Ningxia, China) were tested in research packaging material experiments. Grapes of similar size and quality were tightly wrapped with the CH/LS (2%) and CH (2%) films and incubated at a constant temperature of 18 ◦C in an incubator with relative humidity of 68% to observe the surface changes, compared with those wrapped in polyethylene (20 cm × 20 cm) [29].

### 2.2.3. Determination of Decay Rates

Decay rates refer to the ratio of the weight of rotten fruit to the total weight of treated fruit [30]. The main observations related to whether the appearance of the fruit is full, whether it has edible value, and whether it is corrupted bacterial infection and deterioration. The decay rates of grapes were then calculated according to Equation (1):

Decay rates (%) = number of rotten fruits (N)/total number of fruits (N0) × 100% (1)

### 2.2.4. Hardness

The hardness was checked using a fruit hardness tester (GY-1, JC Group Co., Ltd., Qingdao, China). Before measurement, the hardness tester was placed perpendicular to the surface of the grapes (without breaking the skin), the indenter was pressed into the fruit evenly, and the reading indicated when the pointer stops moving was the hardness value of the fruit [31].
