Beneficial effect and mechanism of walnut oligopeptide on Lactobacillus plantarum Z7

Abstract Prebiotics can stimulate the growth and activity of probiotics and have a variety of physiological functions. However, the study of walnut oligopeptides as prebiotics to promote probiotics is rarely reported. Therefore, in order to explore the beneficial effect of walnut oligopeptide (WOPs) on Lactobacillus plantarum Z7, WOPs was added to the medium of L. plantarum Z7, and the utilization of WOPs, the effect of WOPs on the biofilm, extracellular polymeric substances, and bacterial death were explored. The results showed that the growth‐promoting effect was strengthened with the increasing concentrations of WOPs. The content of bacterial biofilm and EPS increased significantly, and the number of dead bacteria decreased. The beneficial effect of WOPs was probably because that it enhanced the secretion of biofilm which was regulated by bacterial quorum sensing system and promoted the ability of bacteria to resist the adverse environment, thus promoting the growth and reproduction of bacteria.


| INTRODUC TI ON
Probiotics are a kind of bacteria that are colonized in human intestinal tract and reproductive system, which can improve the micro ecological balance and play a beneficial role in human body (Sanders et al., 2013). It is not only a kind of food supplement, but also has been gained more and more attention to its nutrition, health care, and therapeutic effect. In recent years, probiotics have been found to play an important role in the treatment of dermatitis, asthma, gout, diabetes, hepatogenic encephalopathy, gastrointestinal infection, and other diseases (Campaniello et al., 2015;Cordina et al., 2011;Delley et al., 2015;Reid, 2015). However, the number of viable bacteria decreased rapidly in the process of processing, transportation, storage, sales, and consumption. External factors such as temperature, substrate, and oxygen can affect the activity of the strains. Some probiotics have high requirements for nutritional conditions and poor resistance to low pH environment. They are extremely sensitive to oxygen and are difficult to maintain their activity (Doleyres & Lacroix, 2005). In addition, probiotics need to pass through the stomach environment after eating, but a large number of active probiotics died due to the bactericidal effect of gastric acid.
This also reduces the number of viable bacteria in many commercial probiotic products to invalid amount, which can not achieve the beneficial effect on human body. It is an effective way to improve the survival rate of probiotics by adding growth-promoting factor (prebiotic) to probiotics. The prebiotics widely used now include inulin, fructooligosaccharide, galactooligosaccharide, and dextran, but these fast-fermenting oligosaccharides have many disadvantages, | 673 DING aND LI which can cause human diarrhea and flatulence (Puupponen-Pimiä et al., 2002). Therefore, it is of great significance to find and explore the substances and methods that have beneficial effects on probiotics and improve their resistance to high temperature, oxygen, gastric acid, bile, and other adverse environmental factors, which will enhance the maintenance of human physiological health.
Lactobacillus plantarum belongs to probiotic family, which has many functions, such as regulating intestinal flora, regulating blood lipid, enhancing the anti-oxidation ability of the body, participating in the immune response, and reducing cholesterol level. It has also been widely used in the food industry (Behera et al., 2018). Previous studies have shown that the addition of bioactive peptides to probiotics can affect the fermentation process and promote the growth and reproduction of microorganisms (Cudennec & Ravallec, 2013;Yu et al., 2017). Bioactive peptide is a compound composed of two or more amino acid molecules linked by peptide bond, which can participate in physiological activities and play an important physiological role in human body. Some peptides have physical and chemical properties (good cellular diffusion and permeability, small size, low toxicity/side-effects, etc.) and special physiological activities that some proteins do not have, such as promoting fermentation, lowering blood pressure, and anti-oxidation. Thus, bioactive peptides have obtained a great degree of attention and interest. Compared with the same concentration of amino acids, it is easier to be absorbed by intestinal tract. Walnut oligopeptide (WOP) is a kind of bioactive peptide, which has many bioactivities, such as lipid-lowering, anti-oxidation, and antifatigue (Robbins et al., 2012), but its beneficial effect on probiotics has been rarely studied. Therefore, the beneficial effect and mechanism of walnut oligopeptide on probiotics L. plantarum Z7 were studied to provide reference for improving the survival rate of probiotics and the stability of commercial products.

| MATERIAL S AND ME THODS
Lactobacillus plantarum Z7 was stored in our laboratory. The strain (1%) was cultured in the sterilized MRS broth medium for 24 hr (37°C, 160 rpm). MRS broth medium was purchased from Beijing Aoboxing Biotechnology Co., Ltd. Walnut oligopeptide is a light yellow solid powder, mainly composed of small oligopeptide with molecular weight less than 1,000, which contains more glutamic acid, aspartic acid, arginine, and leucine. It was provided by Beijing Tianpeptide Biotechnology Co., Ltd.

| Functional properties of WOPs on the growth of Lactobacillus plantarum Z7
The MRS liquid culture medium was divided into 20-ml anaerobic tubes and sterilized for 15 min (121°C). Then, 0.5%, 1.0%, 1.5%, 2.0% WOPs, and 1% L. plantarum were added into the MRS liquid culture medium, respectively. MRS medium was taken as blank control, and fructooligosaccharide (2.0%) was taken as control. The tubes were cultured at 37°C for 24 hr (160 rpm). Samples were taken every hour to determinate the changes in the number of bacteria. The determination was carried out by using double broth dilution method.
Three appropriate dilution were added to MRS solid medium (1.5% agar was added into MRS liquid medium) and mixed. Then, the plates were incubated at 37°C for 48 hr to count the number of colonies and drew the bacterial growth curve. Every dilution was repeated for 3 times.  (Zwietering et al., 1990). The Modified Gompertz model and Modified Logistic model were used to describe the change rule of L. plantarum Z7 with different concentrations of walnut oligopeptides by nonlinear fitting.

Modified Gompertz model:
The equation of Modified Gompertz model is: In the equation, t is the time (hr); N(t) is the number of bacteria at time t; N max and N 0 are the maximum and initial number of bacteria, respectively (cfu/ml); μ max is the maximum specific growth rate (h −1 ); and λ is the growth lag phase (h).

Modified Logistic model
The equation of Modified Logistic model is: In the equation, t is the time (hr); N(t) is the number of bacteria at time t (cfu/ml); N 0 is the initial number of bacteria (cfu/ml); A is the fitting parameter; μ max is the maximum specific growth rate (h −1 ); and λ is the lag phase (h).

| Utilization of WOPs by Lactobacillus plantarum Z7
Fluorescein isothiocyanate (8.6 mg) was dissolved in 0.2 ml of 0.1 M KOH solution and mixed. Then, 1.6 ml of 0.1 M carbonate buffer (Na 2 CO 3 /NaHCO 3 ) with pH of 8.3 was added. WOPs solution (0.5%, 1 ml) was added to the above solution and mixed.
MRS broth with 200 μl bacterial culture was used as control. Sterile cover glass was put into the dishes and cultured at 37°C for 48 hr.
After culture, the cover glass was washed for 3 times with ultrapure water and dyed with 0.4% crystal violet for 20 min. The bacterial biofilm was observed under oil microscope.

| Effect of WOPs on EPS of Lactobacillus plantarum Z7
2.5.1 | Scanning electron microscope observation of EPS The zinc as described above was took out and washed slowly with sterile water for three times to remove the free bacteria. Then, it was dried for 30 min and immersed in 2%-5% glutaraldehyde (v/v) for 4 hr. Then, the zinc was immersed in 50%, 70%, 80%, and 90% (v/v) ethanol for 10 min and 100% ethanol for twice (15 min each), and finally, it was immersed in 25% isoamyl hexanoate for twice (15 min each). After drying, the zinc was treated by spraying gold and tested by scanning electron microscope (SEM).

| Determination of the chemical composition of EPS
The change of chemical composition of EPS was measured by Raman spectroscopy (HORIBA Scientific Company). The zinc as described above was washed with PBS buffer to remove the free bacteria and dried for 30 min. Then, it was tested by Raman spectrum. Laser source wavelength: 532 nm; laser source power: 25 mW; 50 times objective lens; 5 times of exposure; detection spectrum range: 100-3,500 cm −1 .

| Effect of WOPs on bacterial death of Lactobacillus plantarum Z7
Lactobacillus plantarum Z7 (1%) was cultured in MRS broth medium for 24 hr (37°C, 160 rpm). Then, 100 µl bacterial culture, 10 ml MRS broth medium, and 2.0% WOPs were added to 20 ml anaerobic tubes. Moreover, 10 ml MRS broth with 100 µl bacterial culture was used as control. The tubes were cultured for 24 hr (37°C, 160 rpm), and then, the bacterial culture was washed with normal saline and centrifuged for 5 min (2146.56 g). Then, it was fixed by 70% alcohol overnight at 4°C and washed twice with PBS. Subsequently, it was stained with PI staining kit at 4°C for 30 min, and the unstained L. plantarum Z7 was used as negative control. The bacteria was filtered with 300 mesh nylon mesh and detected by flow cytometry (Cytoflex; Beckman Company).

| Statistical analysis
The results are expressed as the mean ± standard deviation (SD) and were analyzed through one-way ANOVA by SPSS Statistics 16.0 (SPSS, Inc.) Significant differences are displayed at p < .05. All tests were repeated three times.

| Effect of WOPs on the growth of Lactobacillus plantarum Z7
The to the control group. But after 18 hr, the bacteria also entered the decline period. However, the stimulating effect of the culture medium with WOPs was better than that of FOS. The number of living bacteria in the experimental group was significantly higher than that of control group, and the higher the concentration of WOPs, the better the stimulating effect. The logarithmic period of bacterial growth was advanced, and the stable period was also prolonged. During the stable period, the number of living bacteria in the medium supplemented with 0.5%, 1.0%, 1.5%, and 2.0% WOPs reached 3.02 × 10 9 , 4.57 × 10 9 , 1.95 × 10 10 , and 2.69 × 10 10 cfu/ml, respectively. The results showed that WOPs could enhance the metabolic rate of bacteria and promote the proliferation of probiotics.
The growth and change of L. plantarum Z7 were studied by using λ was more obvious than that of FOS. It could be concluded from the above results that WOPs could promote bacterial proliferation and make them enter the logarithmic growth period ahead of time.
WOPs can be used as a growth-promoting factor of probiotics, which will have great application value.

| Utilization of WOPs by Lactobacillus plantarum Z7
The

| Effect of WOPs on biofilm of Lactobacillus plantarum Z7
It could be seen from the quantitative determination of WOPs on the biofilm of L. plantarum Z7 in Figure 3 that both FOS and WOPs could promote the production of bacterial biofilm, but the promotion of WOPs was more significant than that of FOS. Moreover, the higher the concentration of WOPs, the more significant the promotion effect. The content of bacterial biofilm increased by 30.0%, 45.71%, 61.43%, and 84.29%, respectively, when the bacterial culture medium was added with 0.5%, 1.0%, 1.5%, and 2.0% concentrations of WOPs. The quantitative results showed that WOPs significantly promoted the production of biofilm of L. plantarum Z7.
It could be seen from Figure 4 that the biofilm formed by bacteria was dyed by crystal violet (Figure 4a,b). The bacterial distribution of L. plantarum Z7 was relatively scattered when the culture medium was not supplemented with WOPs. However, the cells gathered into groups and formed a large and dense biofilm when the WOPs was added. Moreover, the bacteria wrapped in the biofilm and formed a complex three-dimensional structure. The thickness of biofilm was observed by CLSM (Figure 4c,d). It was found that the thickness of

| Effect of WOPs on EPS of Lactobacillus plantarum Z7
The effect of WOPs on EPS production of L. plantarum Z7 was observed by SEM. It was found that EPS produced by bacteria was little when the culture medium was not supplemented with WOPs  Table S2.
By comparing the corresponding substances of the characteristic peaks of Raman spectrum, it could be seen that the main components of the EPS produced by L. plantarum Z7 were protein, carbohydrate, nucleic acid, and lipid. The Raman spectrum showed that the corresponding substances of EPS in control group in the 720-730 cm −1 and 810-820 cm −1 were mainly adenine, tryptophan, and nucleic acid.

| Effect of WOPs on bacterial death of Lactobacillus plantarum Z7
Flow cytometry can be used to count bacteria, which can quickly and sensitively determine the death of bacteria. This method needs few samples and is easy to operate, which makes it one of the best methods to detect the dead cells at present. From the scatter diagram analysis (Figure 6a,c,e), the forward scatter (FSC) reflected the size and activity of bacteria. The more the x-axis to the right, the larger the cell volume; the vertical axis reflected the particle size and structural changes of bacterial cells, and the more upward, the more complex the refractive substances in cells.
Because the total events of the scatter plot are easy to make a general statistics of the cell fragments, which leads to the inaccurate bacterial count, however, through the P1 circle gate count, the bacteria with uniform morphology can be counted, and the impact of bacterial fragments and adhesion bacteria can be excluded to the greatest extent; thus, the bacterial count data are more accurate. It could be seen from the scatter diagram that after the addition of WOPs, the volume of bacteria and the internal particle size increased, which may be due to the increase of bacterial biofilm and EPS. After the addition of WOPs, bacteria agglomerated and formed a complex three-dimensional structure, which made the total volume and internal particle size of bacteria   (Doleyres & Lacroix, 2005;Hoover, 1993;Ranadheera et al., 2010;Ross et al., 2005;Shah, 2000). Therefore, the proliferation and culture of probiotics have become more and more important. It has been reported that the growth and activity of microorganisms could be stimulated by the addition of exogenous peptide substances. Some enzymatic hydrolysis of proteins from animal and plant sources for the production of bioactive peptides has been taken general attention. Arakawa et al. evaluated the demand of peptide for the growth of Lactobacillus gasseri and evaluated the efficacy of peptide release by enzymatic proteolysis on growth of L. gassei in milk. They found that peptide could be used as a high-quality milk-derived nitrogen source to promote the growth of probiotics, and the growth of probiotics required peptide from milk source, rather than free amino acids or proteins (Arakawa et al., 2015). Dave et al. found that protein hydrolysate could accelerate the growth and reproduction of probiotics in the fermentation process (Dave & Shah, 1997 Bulgaricus (Zhang et al., 2011).

| D ISCUSS I ON
Walnut can be used as medicine and food with a long history. It has high nutritional value and is rich in unsaturated fatty acids, protein, vitamins, folic acid, polyphenols, and other active ingredients (Sze-Tao & Sathe, 2000). WOP is a kind of bioactive peptide, which is obtained from walnut by enzymolysis. It was found that WOPs showed a variety of physiological activities (Li et al., 2017(Li et al., , 2018Liao et al., 2016), but there was little research on its beneficial effect on probiotics. Therefore, it is feasible to explore the stimulation effect of WOPs on the growth of probiotics and its long-term effect as a new prebiotic, which will have great application potential.
When WOPs were added to the culture medium of L. plantarum Z7, the growth rate of bacteria was accelerated, the logarithmic period was advanced, the stable period was prolonged, and the death rate was decreased. These results indicated that the WOPs enhanced the metabolism rate of bacteria, which may be due to that the WOPs improved the tolerance of bacteria to adverse environment. Similarly, Madureira and coworkers also found that probiotic whey cheese promoted the tolerance of Lactobacillus casei, Lactobacillus acidophilus, and Bifidobacterium animalis to simulated digestive tract (Madureira et al., 2011). Extracellular polymer is a kind of polymer secreted by bacteria in certain environment. Bacteria are wrapped in these polymers to form complex biofilm structures, through which bacteria can resist the attack of fungicides and other toxic substances (Danese et al., 2000;Hall-Stoodley et al., 2004). The production of biofilm and EPS is regulated by quorum sensing (QS) system (Liu et al., 2007;Martins et al., 2014). QS is a communication mechanism between bacteria, and it is also a process in which bacteria adjust their physiological and biochemical characteristics according to their population density. Bacteria can produce, release, and recognize extracellular signal molecules called autoinductors (AIs). AIs will accumulate with the increase of bacterial density, and once the threshold concentration of population density is reached, the signal molecules will be sensed by a variety of receptors and then activate or inhibit the expression of specific target genes, leading to a variety of bacterial colony behaviors, such as biofilm production, extracellular enzyme secretion, plasmid transfer, and antibiotic synthesis (Ahumedo et al., 2010;Kim et al., 2015;Wang et al., 2015). It has been proved that probiotics in biofilm state have more significant immunomodulatory effect than probiotics in planktonic state (Rieu et al., 2014). Cheow et al. found that probiotics in high-density biofilm state have better freeze-drying resistance, heat resistance, and acid resistance compared with conventional probiotics (Cheow et al., 2014). Chew and coworkers found that the probiotics Lactobacillus rhamnosus GR-1 and Lactobacillus reueri RC-14 had the ability to inhibit or interfere with the formation of the biofilm of the pathogenic bacteria Candida glabra (Chew et al., 2015).
Vuotto et al. found that probiotics could be used to fight diseases related to biofilm infectious (Vuotto et al., 2014). Therefore, the biofilm of L. plantarum Z7 thickened and EPS secretion increased after the addition of WOPs. The beneficial effect of WOPs was probably due to that it promoted the production of biofilm and EPS which was regulated by QS system, thus increased bacterial resistance to adverse environment. The formation of probiotic biofilm is affected by many factors. It has been reported that the lack of nutrients in the culture medium and the restriction of the supply of carbon source nutrients would promote the formation of biofilm of Lactobacillus rhamnosus GG (Lebeer et al., 2007). Slizova et al. found that the content and types of Tween-80 and sugar in the culture medium played an important role in the formation of biofilm of Lactobacillus reuteri (Slizova et al., 2015). Therefore, the addition of WOPs can be used to improve the formation of biofilm of L. plantarum Z7, which has guiding significance for improving the activity of probiotics.

| CON CLUS ION
This study investigated the effects of WOPs on the growth curve, biofilm formation, EPS production, and death rate of L. plantarum Z7. It was found that WOPs not only promoted the growth and reproduction of bacteria, but also reduced the number of dead cells.
The secretion of bacterial biofilm and EPS increased significantly after the addition of WOPs, so the promoting effect of WOPs on the growth of L. plantarum Z7 was probably related to the quorum sensing system of bacteria. WOPs can be used as a new and potential prebiotic to promote the growth and quality nutritional value of related products.

ACK N OWLED G M ENTS
This research received no external funding.

CO N FLI C T O F I NTE R E S T
All authors declare no conflict of interest.

E TH I C A L A PPROVA L
This study does not involve any human or animal testing.

I N FO R M E D CO N S E NT
Written informed consent was obtained from all study participants.