Application of surface-active substances produced by Nocardia vaccinii ІМВ В-7405 for the treatment of vegetables

Introduction. Application of surface-active substances (SAS) produced by Nocardia vaccinii ІМV В-7405 for shelf live extension of vegetables was studied. Materials and methods. Organic vegetables such as tomato, cucumbers, and squashes were washed with the solution of SAS produced by N. vaccinii ІМV В-7405 with concentrations of 0.25 or 0.5 g/L. Microbiological analysis was done before the beginning of the vegetables storage. Evaluation of vegetable quality was conducted by viewing during time of the storage. Results and discussion. The results of our research showed the efficiency of the application of biosurfactant produced by Nocardia vaccinii ІМV В-7405 using industrial wastes for shelf life extension of vegetables. Results of visible observations as well as microbiological analysis showed that the treatment of vegetables with SAS solutions at the concentrations of 0.25 and 0.5 g/L was more effective than washing them with tap water. The total number of heterotrophic bacteria and fungi in the samples decreased after treatment of vegetables with SAS of N. vaccinii ІMV B-7405 by 16–34 and 3–14 times, respectively, meanwhile the washing of vegetables with tap water decreased total microbial number only by 2–2.5 times. It was shown that vegetables washed with water spoiled faster than those treated with SAS solution. The advantages of application of this biosurfactant for vegetables post-harvest treatment are that (1) it can be used at the lower by 2–6 times concentration in comparison


Introduction
Shelf life of fresh vegetables is a matter of great importance. According to statistics of the Food and Agriculture Organization of the United Nations (FAOSTAT), the developed countries lose only 5% of the harvest due to the more perfect technologies of vegetables storage, meanwhile in the other countries the losses of harvest can be above 20% [1,2]. For example, in China the losses of 30 million tons of annual garden-stuffs economic damages can be estimated as 30 billion yuan [1][2][3]. Ukraine is one of the leading world producers of vegetables. However, the total post-harvest losses were assessed as 800-964 thousand tons in 2017 [4], which consist about US$30-37 million. To extend shelf life of vegetables they can be treated using physical methods such as hot-water immersion, low temperature storage, modified atmosphere packaging technology, the treatment with gamma radiation or by pulsed electric field and chemical methods such as application of acidifying agents, chemical reductants, inhibitors of enzymes activities, sequestrants [5][6][7][8]. The treatment of the vegetables using physical methods can cause their mechanical damages such as bruises, abrasions and cuts which make them more sensitive to desiccation and shriveling, increase in respiration rate and ethylene emission, speed up the wilting and enzymatic browning. The high temperatures can cause yellow discoloration of green vegetables, and the use of modified atmosphere packing stipulates determination of optimal gas composition for every certain product [8]. Application of the physical methods for vegetables treatment also requires special equipment.
Application of synthetic chemical substances for surface treatment of vegetables is not appreciated by consumers because of the health concerns. For example, application of such widely used as a post-harvest dip of crops synthetic antioxidants as ethoxyquin and diphenylamine are under regulatory restrictions [7]. So, the global trend is to decrease the use of the synthetic chemical substance for prolongation of the vegetables shell life. To do it, the development of alternative safe methods is needed. The antimicrobial and antiadhesion efficiency of nontoxic biodegradable microbial surface-active substances (SAS), for example, sophrolipids and rhamnolipids, to treat the agricultural products was shown [9][10][11][12][13]. However, this treatment is costly. To diminish the cost of the post-harvest treatment of vegetables with microbial polysaccharides the strain producing surface active substances that are by one order of magnitude more active than known substances was selected and studied. The strain of hydrocarbon-oxidizing bacteria Nocardia vaccinii ІМV В-7405 isolated from the oil-polluted soil was able to synthesize substances, which had surface activity and emulsion properties [13]. It was shown that this biosurfactants had also antimicrobial and antiadhesive properties [14,15] and was active against phytopathogenic bacteria of the geniuses Pseudomonas and Xanthomonas [16].
The aim of the present research was testing of the surface active substances produced by N. vaccinii ІMV B-7405 for the vegetables treatment to extent their shelf life.

Microorganism
The strain Nocardia vaccinii ІМV В-7405 was isolated from the oil-polluted soil [13] and was deposited in the Collection of Microorganisms of Institute of Microbiology and Virology NAS of Ukraine. The biosurfactant produced by N. vaccinii ІМV В-7405 was a set of neutral, glyco-and aminolipids. Neutral lipids contained mycolic and n-alkanoic acids, glycolipids contained trehalose diacelates and trehalose mycolates [14].

Cultivation of N. vaccinii ІМV В-7405
Bacterial strain N. vaccinii ІМV В-7405 was cultivated in the liquid mineral medium with the following composition, g/L: NaNO3, 0.5; MgSO4•7H2O, 0.1; СaCl•2H2O, 0.1; KH2PO4, 0.1; FeSO4•7H2O, 0.001, yeasts extract, 0.5 vol%, distilled water 1 L. Crude glycerol which is waste from biodiesel production plant, Poltava region, Ukraine, was used as a source of carbon in the concentration of 2 vol.%. Inoculum was prepared by cultivation of the strain N. vaccinii ІМВ В-7405 in the medium described above with 0.5 vol.%. of crude glycerol. The cultural liquid with the concentration of bacterial cells 10 4 -10 5 cells/mL was taken from the exponential growth phase and was added to the medium for the strain cultivation, 10 vol.%. Cultivation of N. vaсcinii ІМV В-7405 was conducted in the flasks with the volume of 750 mL in 100 mL of medium under shaking at 320 rpm at 30 ºС during 120 hours.

Production of surface-active substances
The cultural liquid after cultivation was centrifuged at 5000×g for 45 minutes (laboratory centrifuge LP-8, Kiev, Ukraine). The Folch solution (chloroform and methanol in volume ratio 2:1) was used for extraction of surface-active substances. The supernatant was placed in the funnel, then Folch solution was added (ratio 1:1), and the mixture was shaken for 5 min and left for phase separation. The lower fraction (organic extract 1) was removed, and the extraction procedure was repeated twice to obtain organic extracts 2 and 3. All extracts were combined and evaporated on the rotor evaporator ER-1M2 (Russia) at the temperature 50 о С and pressure 0.4 atm to the constant weight. The dry SAS was dissolved in water and solution of biosurfactant with its concentration of 0.25 -0.5 g/L was used in the research.

Vegetable washing
To study the influence of the vegetable treatment with SAS of N. vacсinii ІМV В-7405 on the storage duration, picked up vegetables were divided into three groups with 10 -30 pieces in each. Vegetables of the first group were not treated at all, vegetables of the second group were washed with tap water, and vegetables of the third group were washed with the solutions of SAS with concentrations of 0.25 or 0.5 g/L. Vegetables were placed in the glass cylinder, 250 mL of tap water or the SAS solution was added, treatment lasted for 5 min, and after that vegetables were taken off and placed on the plates at the room temperature for observation [8]. Microbiological analysis was done before the beginning of the vegetables storage.

Microbiological analysis
Some vegetables from each group were taken with sterile pincers, then they were placed into sterile porcelain jar and were pestling. Homogenized mixture, 10 g, were placed into flask with 90 mL of sterile tap water and shaken. The quantity of microbial cells (colonyforming units, CFU) was determined by the plate diluting method. The quantity of heterotrophic bacteria was determined by their growth on the beef-extract agar-agar (BEAA) at 37 ºС for 24 hours, and the quantity of the fungi was determined by their growth on the Wort Agar for microbiology at 30 ºС for 48 hours.

Evaluation of vegetable quality
Evaluation of vegetable quality was conducted by viewing during time of the storage. The experiment was finished when the signs of deterioration (usually on the seventh day) such as decay, changes of color and texture, the presence of the cracks and wrinkling were evident on all vegetables.

Statistical analysis
All experiments were done in triplicates, the number of the parallel determinations varied from 3 to 5. Statistical analysis was done using computer program Statistix 10.0 for Windows version 11.5. The average means (M) and standard deviations (SD) were calculated for the experimental results.

Results and discussion
From the different vegetables, which are cultivated in Ukraine, the cucumbers, the squashes, and the tomatoes were chosen for our experiments by the following reasons: (1) according to the data of FAOSTAT Ukraine occupies sixth place in the world on the cucumbers production going ahead USA and Spain and is within the top twenty exporting countries [17]; Ukraine is a leader in Europe in the production of tomatoes, and the part of tomato's production contributes 20%. of overall fruit-and-vegetable production of the country [18]. In addition, tomatoes unlike vegetable marrows and cucumbers belong to the soft garden-stuffs, so at the longtime transportation or storage they are quickly infected by microorganisms and spoiled.
In the studies of the influence of microbial SAS on the extension of the storage time of fruit-and-vegetables products high enough concentration of preparations, 1 -3 g/L, were used [2,8,[18][19][20][21]. Our previous studies showed that SAS synthesized by N.vacсinii ІМV В-7405 had high antimicrobial activities [16] [23]. Therefore, in our present study the lower concentration of SAS produced by N.vacсinii ІМV В-7405 (0.25-0.5 g/L) and used in experiments was comparable with the concentrations reported in the literature (1-3 g/L).
Visual supervision of the cucumbers and the squashes shown that the samples treated with SAS did not have the signs of spoilage after 7 days of storage in comparison with ones washed with water ( Figure 1).  According to the visual observation, external appearance of tomatoes treated with the SAS solutions with concentration 0.25 or 0.5% on the seventh day was almost the same ( Figure 5). Taking into account the economic consideration, dosage of SAS 0.25 g/L was recommended for the treatment of vegetables for extension of their shelf life, notwithstanding that concentration of SAS 0.5 g /L was more effective. Currently, there are known only some studies of applications of microbial surfactants for fruit and vegetables treatment, and the industrial production of these surfactants (rhamnoand sophorolipids) is conducted by only few companies, the most of which work in the USA [24]. American companies AGAE Technologies LLC, Jeneil Biosurfactant Co. LLC, Paradigm Biomedical Inc., Rhamnolipid Companies Inc. produce rhamnolipids to be used in pharmaceuticals industry, agriculture, for bioremediation of polluted environment, and to increase of oil production. Japanese companies Saraya Co. Ltd. and Allied Carbon Solutions (ACS) LTD Japan produce sophorolipids, which are used in the manufacturing of cleaning and hygiene products, as well as in agriculture.
Low yield of biosynthesized SAS and their high production cost are the major restrictions for their wide use. One of the ways to diminish the cost of biosurfactant production is the use of the waste materials as substrates for SAS biosynthesis [25], for example, wastes from fat-and-oil, sugar, and milk industries, agriculture and forestry, production of biodiesel, and used (overdone) oils. The most suitable substances for the biosynthesis of the microbial surfactants are oil-containing wastes, which unlike lignin-and cellulose-containing wastes, milk whey, and technical glycerin, do not need preliminary treatment and purification.
It was shown in our previous studies that the wastes from the biodiesel production and used sunflower oil can serve as the substrates for cultivation of N.vacсinii ІМВ В-7405 and synthesis of biosurfactant [14,26]. Additionally, this biosurfactant can be used for the vegetables treatment in the concentrations much lower than ones that are reported in the literature. So, sophorolipids produced by Wickerhamiella domercqiae Y2a CGMCC3798 were used to treat citruses, peaches and apricots in the concentrations of 1-3 g/L [2]. It was shown that the strongest hyphae growth spread of Aspergillus niger, Aspergillus flavus and Penicillium (agent of fruits putrefaction) was suppressed at concentration of the sophorolipids fruit preservative of 3 g/L [2]. Inhibiting effect of the treatment of Eugenia uniflora (Surinam cherry, pitanga) with rhamnolipids solution produced by Pseudomonas aeruginosa LBI in the concentration of 1 g/L was shown [7]. It is known that biosurfactants can be used at the lower concentrations but only in combination with other components, for example, with synthetic SAS or with microorganisms, which are antagonists of phytopathogens. So, Yan with coauthors showed the synergetic effect of rhamnolipids of Pseudomonas aeruginosa (0.5 g/L) and suspension of the yeasts Rhodotorula glutinis (1×10 8 cells/mL) in suppressing Alternaria alternata on cherry tomato [19]. A simultaneous application of R. glutinis and rhamnolipids to treat the tomato surface to control post-harvest diseases was more effective than application of yeasts and biosurfactants alone. Application of 0.25% solution of sophorolipids, synthesized by Candida bombicola ATCC 22214, in combination with synthetic SAS polyethylene glycol was effective against Erwinia chrysanthemi ATCC 11663 and Xanthomonas campestris ATCC 13951 on the surface of chikoos, tomatoes, cucumbers, and citruses [22].

Conclusions
The results of our research showed the efficiency of the application of biosurfactant produced by Nocardia vaccinii ІМV В-7405 for shelf life extension of vegetables. The advantages of application of this biosurfactant for vegetables post-harvest treatment are that (1) it can be used at the lower by 2-6 times concentration in comparison with other reported in literature microbial SAS, and (2) it can be produced using industrial wastes that will reduce the cost of its production. So, use of biosurfactant synthesized by N.vacсinii ІМВ В-7405, which will be produced using industrial wastes, can resolve such important problems as utilization of the toxic industrial wastes, decrease of the cost of biodiesel production, and extend the shelf life of vegetables during their storage and transportation.