Beneficial effectiveness of probiotic-low-fat ice cream containing

F U L L P A P E R Beneficial effectiveness of probiotic-low-fat ice cream containing Krueo Ma Noy (Cissampelos pareira L.) gum on colon microbiome under a dynamic gut model Kemsawasd, V. and Chaikham, P. Institute of Nutrition, Mahidol University, Nakorn Pathom campus, Nakorn Pathom 73170, Thailand Division of Food Science and Technology, Faculty of Science and Technology, Phranakhon Si Ayutthaya Rajabhat University, Phranakhon Si Ayutthaya 13000, Thailand


Introduction
Functional foods, also known as foods exerting beneficial effects on specific organs or systems within the human body, are known for a variety of functions beyond energy and nutrient provision (Granato et al., 2010).In the present day, growing numbers of healthconcerned consumers fueled the rapid expansion of functional foods in the market, especially probiotic dairy products.In terms of functional foods, dairy products are strongly linked to probiotics.Probiotic strains have become of prominent interest in food product development due to its various properties, such as bacteriocin production, acid and bile tolerance, epithelial cell adherence, gut survivability and colonization, and endurance to physiochemical conditions of food processing and storage, which contribute to numerous benefits on human health (Prado et al., 2008).Such benefits are attributed by their antimicrobial, antimutagenic, anticarcinogenic and antihypertension properties.Regular ingestion has been shown to reduce symptoms of food allergy, stabilize host gut microflora and stimulate gut resistance to pathogens (Liong et al., 2009;Granato et al., 2010).
Dairy products, viz.probiotic yogurts, fermented beverages and ice cream, are regarded as the main vehicles of probiotic supplementation and subsequent ingestion.According to Shah (2007), at least 10 6 CFU/g of probiotic cells should be present in a dairy food to compensate subsequent population reduction during passage along the human gut.Probiotic supplemented ice cream has been reported to promote promising health benefits (Çaglar et al., 2008) and may potentially serve as vehicles and protective barriers for probiotics during gut transport (Cruz et al., 2009).The prevalence of noncommunicable diseases, such as obesity and cardiovascular disease, has led to towards the innovation and development of low-fat ice cream formulations containing probiotic cultures, prebiotics and synbiotics (Akalin and Erisir, 2008;Chaikham and Rattanasena, 2017).
Krueo Ma Noy (Cissampelos pareira L.) is a woody, climbing medicinal plant of the Menispermaceae family indigenous to the tropical area of Asia, East Africa and South Africa.This plant grows abundantly in the Northern and Northeastern regions of Thailand.Due to its analgesic properties, the Krueo Ma Noy plant was commonly used by indigenous people to treat a number of ailments, including sores, asthma, dysentery, diuretic and traumatic pain (Vardhanaburi and Ikeda, 2006).Cold -water extraction of the leaves produced a dark green gel with cooling properties that can be used to treat fever.Gel formation occurred within a very short time after the extraction process, resulting from unique characteristics of Krueo Ma Noy pectin, a complex polysaccharide examined in the reports of Singthong et al. (2004Singthong et al. ( , 2005)).Pectin is commonly applied in food production and manufacturing processes as gelling and thickening agents (Barros et al., 2002).The water-soluble fiber is one of the major substrates in the colonic bacteria metabolism of carbohydrate hydrolysis to produce organic byproducts, such as short-chain fatty acids (SCFA) and lactic acid (Slavin, 2013).Citrus pectin hydrolysate has been shown to exhibit prebiotic effects on tested non-fat milk probiotics, as demonstrated by growth enhancement, increased fermentation, and high levels of probiotic survivability (Ho et al., 2017).
Many researchers have incorporated dynamic models of the gastrointestinal system in the study of various probiotic functional foods, prebiotics and prebiotic candidates regarding their effects on colon bacteria viability, microbial composition and metabolite synthesis (Mäkivuokko et al., 2006;Mäkivuokko et al., 2007;Chaikham et al., 2012;Bianchi et al., 2014;Apichartsrangkoon et al., 2015).Different in vitro gut models were used predominantly in nutrition studies involving compositional analysis of the colon microbial community.Normally, they are comprised of different segments of the human gastrointestinal tract, including stomach, small intestinal and colon compartments, with a stable microbial community resembling the human gut microbiota or conditions (Kontula et al., 2002;Possemiers et al., 2010;Sivieri et al., 2013).Probiotic administration has been shown to enhance the modulatory effects of low-fat ice cream on simulated microbial ecosystems of the gastrointestinal tract (Chaikham and Rattanasena, 2017).While the fermentation of different prebiotics and prebiotic candidates on probiotic foods have been subjected to the similar simulation systems, there are currently insufficient studies attributing to the potential prebiotic effects of Krueo Ma Noy gum (KMN).Therefore, the objective of this study was to examine the combined ability of KMN and Bifidobacterium lactis Bb-12 supplementation along with low-fat ice cream in adjusting microbial compositions of the simulated gut model.Colon microbial diversities were determined by monitoring the changes in populations of colon microorganisms, viz.lactobacilli, bifidobacteria, clostridia, fecal coliforms and total anaerobes, after feeding and fermentation with different treatments of low-fat ice cream.The ice cream samples were either administered pure, supplemented with B. lactis Bb-12, KMN or both.Metabolic products of microfloral fermentation, including SCFA (acetic, propionic, and butyric acids), lactic acid, ammonia and biogenic amines (cadaverine, putrescine, methylamine and tyramine) were also assessed within this study.

Extraction of Krueo Ma Noy gum
Krueo Ma Noy leaves were harvested from an orchard in Sakon Nakhon province, Thailand.The washed leaves were sun-dried for 15 hrs before blending.The blended leaves were extracted with distilled water at a ratio of 1:20 (w/v) at 75°C for 1 hr (Singthong et al., 2005).The extract was then filtered and precipitated with absolute ethanol at a ratio of 1:3 (v/v).Afterward, the precipitate was dried using a vacuum oven (XF050, France Etuves, Chelles, France) and powdered using a blender (National, Bangkok, Thailand).

Preparation of probiotic pellet
Lyophilized B. lactis Bb-12 was incubated in sterile MRS broth containing 0.05% (w/v) L-cysteine hydrochloride at 37°C for 20 hrs and then harvested by FULL PAPER centrifugation (Rotina 46R Centrifuge, Hettich ® , Tuttlingen, Germany) with a rotary speed of 4,500 rpm at 4°C for 20 mins.Afterward, the precipitated cells were washed twice with 0.85% (w/v) sterile saline water and kept in a refrigerator at 4°C for 16 hrs (overnight).For activation, the pellet cells were warmed at 37°C for 1 hrs before supplementation into low-fat ice cream.

Gut model experiment
Basal nutrient medium and pancreatic solution were prepared (Table 1) and sterilized at 121°C for 15 mins.The basal nutrient medium was acidified with 37% (conc.)hydrochloric acid to pH 2. The acidified basal nutrient medium and pancreatic solution were then used for feeding into the dynamic gut model with 1 ml/min flow rate.Four sterile fermentation vessels which consisted of the stomach (1 st vessel), small intestine (2 nd vessel), proximal colon (3 rd vessel) and distal colon (4 th vessel) were set up and operated according to the running-setup protocol of Chaikham et al. (2012) with some modifications.The system temperature was set at 37°C, by means of a circulating water bath, and culture pH was maintained at 5.6-5.9 and 6.6-6.9 in proximal and distal colon vessels using a pH controller with the automated addition of 0.25 M hydrochloric acid and 0.1 M sodium hydroxide.Each colon vessel was inoculated with 30 mL of fecal slurry from five healthy donors which were prepared using pre-reduced 0.1 M phosphate buffer saline (pH 7) plus 2 g of sodium thioglycolate (reducing agent) and then mixed in a stomacher for 5 mins.Fecal samples were obtained from three male and two female volunteers with a mean age of 25 years who had not taken antibiotics for at least 6 months prior to providing the sample and had no history of a gastrointestinal disorder.Various treatment compositions were fed continuously into the model for 9 weeks, as shown in Table 2.The basal period (2 weeks) was intended to modulate the microbiome in the different colons to the prevailing conditions in order to obtain a population that resembles the in vivo situation in terms of either community composition or metabolic activity (Chaikham et al., 2016).For collecting the fermented samples in both colon vessels, after 2 days feeding, 20 mL of colon fluids were withdrawn daily for assessments of microflora metabolites and populations.

Determinations of ammonia and biogenic amines
Ammonia concentration in colon fluids was determined according to the method as described by Chaikham et al. (2012).A 1-mL colon fluid was added with one teaspoon of magnesium oxide and the mixture was then distilled using a Kjeldahl Apparatus Vapodest 30 S (Gerhardt, Königswinter, Germany).The released ammonia gas was entrapped with boric acid solution before titration with 0.02 M hydrochloric acid.The ammonia concentration was calculated and expressed as gram ammonia per 1 L colon fluid (g/L).
Cadaverine, putrescine, methylamine and tyramine were determined using the modified HPLC method as described by Tosukhowong et al. (2011).A total of five milliliters of colon fluids were well-mixed with 35 mL of 10% TCA solution before centrifugation at 4,000 rpm for 15 mins.One milliliter supernatant was mixed with 0.2 mL of 2 M sodium hydroxide and 0.3 mL of saturated sodium bicarbonate and then allowed to stand at room temperature for 30 mins.A 2 mL of 10 mg/mL dansyl chloride solution was added into the mixture before incubation at 40°C for 30 mins.After that, 0.1 µL of 25% ammonia was added to stop the reaction and centrifuged at 4,000 rpm for 10 mins.The supernatant was filtered through a 0.20-µm nylon membrane filter and 20 µL filtrate was injected into a Shimadzu HPLC system (CL-10 ADVP, Shimadzu, Kyoto, Japan).Separation of biogenic amines was achieved using a C18 column (YMC-Pack ODS-AM, 5 µm, 4.6 mm ID × 250 mm; YMC, Kyoto Japan).The mobile phase was a mixture of 0.1% acetic acid (solvent A) and 0.1% acetic acid in acetonitrile (solvent B) with a flow rate of 1.0 mL/min.The gradient system of the mobile phase commenced from 0 min (50% A and 50% B) to 30 mins (10% A and 90% B), 10 mins (50% A and 50% B) and maintained at this state to 10 mins.The temperature of the column was set at 40°C and UV detection was at 254 nm.The peak area of each component was determined and converted to concentration.

Statistical analysis
Five replicates were performed, and the mean value was calculated.Analysis of variance (ANOVA) was used to carry out the variation and significance of difference.Determination of significant differences within treatment means was done using Duncan's multiple range tests at 95%.Pearson correlation coefficient was used for grouping the DNA patterns on PCR-DGGE fingerprints.

Short-chain fatty acids and lactic acid
The results in Table 3 showed the concentrations of acetic acid, propionic acid, butyric acid, total SCFA and lactic acid presented in proximal and distal colon vessels following treatments of basal nutrient medium (Table 1) along with low-fat ice cream, B. lactis Bb-12, KMN and combination with both B. lactis Bb-12 and KMN (Table 2).The gut model was left for long-term running with periodical washouts following 7 days of different treatments, as can be observed from Table 2. Regarding SCFA, it was noticed that acetic acid concentrations were of the highest levels compared to propionic and butyric acids in both colon vessels (P≤0.05).This complied to the population data from various studies showing the greatest acetic acid production among the three types of SCFA, where ratios of acetic, propionic and butyric acids production in large intestinal follow an approximate ratio of 3:1:1 (Topping and Clifton, 2001).Probiotic-low-fat ice cream (Treatment 2) also FULL PAPER significantly increased (P≤0.05) the levels of all SCFA compared to pure low-fat ice cream (Treatment 1).Similarly, in the study of Chaikham and Rattanasena (2017) on the effect of low-fat ice cream supplementation with probiotics (Lactobacillus casei 01 and Lactobacillus acidophilus LA5) on human colonic microflora communities, probiotic along with low-fat ice cream was shown to boost the production of SCFA, as demonstrated by the increase of all SCFA (acetate, butyrate and propionate) in both proximal and distal colon vessels of the human colon simulator compared to pure low-fat ice cream.However, the concentration of total SCFA was significantly higher (P≤0.05) in the presence of low-fat ice cream plus KMN (Treatment 3) during fermentation, expressed as 3.36±0.09and 3.80±0.13g/L in respective proximal and distal colon vessels, when compared to treatments of low-fat ice cream alone and the latter in combination with B. lactis Bb-12.
In this case, the simulated colon treatment incorporating the addition of B. lactis Bb-12 supplemented low-fat ice cream together with KMN (Treatment 4), was proven to be the treatment combination that can most effectively modulate the composition of all SCFA studied in this research (P≤0.05).A significant increase in SCFA concentrations was established in proximal and distal colon vessels, of 3.89±0.13and 4.73±0.15g/L, respectively, compared to probiotic-low-fat ice cream, low-fat ice cream with KMN and pure low-fat ice cream.There was currently insufficient research on the synergistic effects of KMN on the human colon microbiome, many dietary fibers, including pectin, have been used as prebiotics to deliver beneficiary effects to alter the gut microbial population and improve host health (Woods and Gorbach, 2001).Pectin is readily soluble in water and initiates gel formation within the gastrointestinal tract, enhancing gut microflora fermentation processes.Gel matrices increase the surface area available for enzymatic reactions and bacteria-mediated degradation processes of undigested food substances (Gibson, 2004).SCFA formed through food and nutrient fermentation by the gut microbiota confer numerous health benefits to the host.SCFA serve as nutrients for colonic epithelium, modulators of pH within intracellular and colon environments, regulators of cell volume and as enhancers of metabolic processes, such as ion transport, cell proliferation and mineral absorption (Cook and Sellin, 1998).There are a number of scientific evidence supporting the roles of SCFA as major regulators of colonic health and are known to exhibit preventative effects against gastrointestinal disorders, cancer, anti-cardiovascular diseases and suppress pathogen growth (Van Immerseel et al., 2010;den Besten et al., 2013).
Here, the impact of various treatments on lactic acid concentration in the human gut reactor was observed in Values are expressed as mean values ± standard deviation (n = 5).Means in the same rows with the same lowercase letters and means in the same columns with the same uppercase letters indicate no significant difference (P > 0.05).
Table 3.While the control treatment (basal period) contained lowest concentrations of 0.54±0.10 and 0.69±0.12g/L lactic acid in both proximal and distal colon vessels, respectively, all treatments in the presence of low-fat ice cream demonstrated a significant increase in lactic acid in both compartments (P≤0.05).Noticeably, while lactic acid concentrations increased with treatments of low-fat ice cream with B. lactis Bb-12, and of low-fat ice cream with KMN, the greatest increase in concentrations of lactic acid observed in both reactor vessels were accounted by treatments of low-fat ice cream in combination with B. lactis Bb-12 and KMN, which were1.74±0.16g/L in proximal colon and 1.95±0.13g/L in distal colon.Some of the factors contributing to higher levels of lactic acid can be accounted by the ingredients of low-fat ice cream, including sucrose and cornstarch, which served as carbohydrate substrates of colon microbiota metabolism, fermentation, and increased synthesis of lactic acid and SCFA.It is known that lactic acid is one of the major end-products of Bifidobacterium fermentation, along with other strains of genera Lactobacillus (Biavati, 2001;Pokusaeva et al., 2011).Thus, the inoculation of probiotics allows for increased substrate utilization to increase the yields such organic acids (Chaikham et al., 2012;Chaikham and Apichartsrangkoon, 2014;Chaikham and Rattanasena, 2017).Although little research has been conducted on the prebiotic properties of KMN, results suggested increased metabolic byproduct yields of the simulated gut model in its presence.
The presence of pectin in KMN may have contributed to this effect.
Comparing the levels of SCFA and lactic acid within the two colon compartments, the concentrations of all organic acids were significantly higher (P≤0.05) for all treatment compositions in the distal colon compared to the proximal colon.Overall, a more significant rise in levels was observed for SCFA as opposed to lactic acid.These results suggested the presence of unabsorbed organic acids, as digestion of the treatments proceed from simulated proximal (ascending and transverse) to distal (descending) colons (Chaikham et al., 2012).Similar trends of increase in acetate, butyrate, propionate and branched acid levels in ascending, transverse and descending colon compartments could be observed in the report of Van de Wiele et al. (2006) on in vitro prebiotic effects of longer-degree polymerized inulin-type fructans.In healthy humans, the production rate of SCFA in the proximal colon was higher than that of the distal colon due to high substrate concentration.However, the SCFA in the distal colon was higher than that of the proximal colon due to the accumulation of SCFA in the colon (MacFarlane et al., 1992).Subsequent depletion is defined by lower levels of SCFA production, from ∼250 mM SCFA/kg fecal/48 hrs in the proximal colon to ∼50 mM SCFA/kg fecal/48 hrs in the distal colon (MacFarlane and Gibson, 1995).While fermentation is predominant in the human proximal colon, trace amounts (~5%) of SCFA unabsorbed by colonocytes are transported and deposited along distal regions, and ultimately expelled via fecal matter excretion (Topping and Clifton, 2001).

Colon microbial populations and PCR-DGCE fingerprints
Enumeration and subsequent colony count determinations of specific bacterial strains and total anaerobes were conducted to observe the changes in in vitro gut microbial compositions under varied treatment compositions of low-fat ice cream, washout period and basal conditions.The colony units were expressed in terms of log CFU/mL colon fluid, as can be observed from Table 4. Compared to basal microbial concentrations, it was found that all treatments of low-fat ice cream (Treatments 2, 3 and 4) in both colon vessels significantly increased (P≤0.05)levels of colonic lactobacilli by approximately~1.3-1.9 log CFU/mL (proximal colon) and ~1.0-1.8 log CFU/ml (distal colon), and of bifidobacteria by ~1.3-2.4 log CFU/mL (proximal colon) and ~1.0-2.0 log CFU/mL (distal colon) as compared to basal treatment (Treatment 1).According to Chaikham and Rattanasena (2017), ingredients of ice cream provide carbon and nitrogen sources, whereas probiotic inoculation increases the utilization capacity of those aforementioned fermentation substrates.
Although supplementation of low-fat ice cream with B. lactis Bb-12, KMN and both in combination significantly increased (P≤0.05)lactobacilli prevalence in the gut reactor compared to pure low-fat ice cream, there was no significant difference (P>0.05) in the quantities of lactobacilli among the supplementation with probiotic or KMN on their own within the proximal colon compartment (~7.9-8.0 log CFU/ml).In the distal colon vessel, however, significant increases were primarily observed in treatments containing B. lactis Bb-12 (Treatments 2 and 4).Combined supplementation of low-fat ice cream, probiotic and KMN yielded the highest distal colon population of beneficial lactobacilli (8.24±0.10log CFU/ml).Similar trends were also observed for bifidobacteria levels in both vessels, which can be accounted for by direct inoculation of B. lactis Bb -12 in the basal nutrient medium.The greater prevalence of lactobacilli and bifidobacteria suggested synergistic effects of B. lactis Bb-12, and potential prebiotic effects of KMN on the growth of beneficial colon bacteria.Similarly, a study by Bianchi et al. (2014) showed FULL PAPER enhanced prevalence and diversity of Lactobacillus sp.communities after feeding prebiotic and probioticsupplemented vegetable beverages into a Simulator of the Human Intestinal Microbial and Ecosystem or SHIME reactor.According to some other previous studies, pectic oligosaccharides and pectin derived from orange and lemon peel, sugar beet pulp and kiwi fruit were observed to have excellent overall and bifidogenic prebiotic properties, as proven by increased probiotic populations and enhanced SCFA formation (Hotchkiss et al., 2003;Parkar et al., 2010;Gómez et al., 2014;Gómez et al., 2016).
Observing from the trends of change in the compositions of the simulated gut microbiome, the results in Table 4 elucidated a significant decrease (P≤0.05) in the populations of fecal coliforms, clostridia and total anaerobes upon treatment with varied compositions of low-fat ice cream compared to basal conditions (control) in proximal and distal colon vessels.While feeding of the gut reactor with low-fat ice cream resulted in the least diminishing effects on harmful bacteria populations, there were no significant differences (P>0.05) between reduced levels of aforementioned bacterium upon separate supplementations of low-fat ice cream with B. lactis Bb-12 and with KMN in proximal and distal colon vessels.Combined supplementation of probiotic and KMN, however, resulted in the greatest reduction in levels of clostridia, total anaerobes and fecal coliforms in both vessels.Such effects following supplementation with KMN (Treatment 3) and the latter in combination with B. lactis Bb-12 (Treatment 4) were not of significant difference (P>0.05).Clear correlations between increased levels of beneficial gut bacteria and organic acids (Table 3) in the simulated gastrointestinal environment can be explained by their immunemodulatory and regulative functions, and natural inhibitory effects of common gut pathogens, including Staphylococcus aureus, Clostridium botulinum, Listeria monocytogenes, and some harmful strains of Escherichia coli (Maslowski and Mackay, 2011;Chaikham et al., 2013).Chaikham et al. (2012) reported that the probiotics or/and prebiotics possibly produced lowmolecular-weight antimicrobial substances (i.e.lactic acid, various SCFA and hydrogen peroxide) to inhibit the pathogenic bacteria.The study of Fooks and Gibson (2003) have shown enhanced antimicrobial effects, specifically inhibition of gut pathogens, E. coli and Campylobacter jejuni, by synbiotic treatments of Lactobacillus plantarum 0407 with oligofructose and Bifidobacterium bifidum with a mixture of oligofructose and xylo-oligosaccharides within an anaerobic fermentation system containing human feces.On a similar context, feeding of mice with probiotic Lactobacillus helveticus M92 and various kinds of prebiotics were shown to increase gastrointestinal populations of lactic acid bacteria, while the reduction in the levels of enterobacteria and sulphite-reducing clostridia were observed (Frece et al., 2009)

Ammonia and biogenic amines
Many strains of colon bacteria utilize urease, a metabolic enzyme, in converting nitrogenous compounds, including protein, peptides, amino acids and urea, into ammonia and biogenic amines (BA).BA are often formed via amino acid decarboxylation present in their biosynthetic pathways by various types of bacteria such as Enterococcus, Escherichia and Morganella strains (Mäkivuokko et al., 2010).Dairy products are prone to contain high levels of BAin the colon compartments due to its rich and balanced chemical composition, providing suitable conditions for microbial growth (Linares et al., 2011;Benkerroum, 2016).Table 5 portrays ammonia and BA formation in the colon vessels prior to and after low-fat ice cream addition.Evidently, basal concentrations of toxic nitrogenous compounds were lowest compared to low-fat ice cream treatments (P≤0.05) in both colon vessels.Highest levels of ammonia and BA accumulation were observed in the treatment of pure low-fat ice cream (Treatment 1), while cadaverine was presented at highest levels compared to other specified BA for all treatments in both colon vessels.Pure low-fat ice cream contained skim milk and gelatin, thus may serve as major protein sources for gut bacteria in the metabolic process of substrate utilization.
Following separate treatments of low-fat ice cream with B. lactis Bb-12 (Treatment 2) and with KMN (Treatment 3), levels of toxic metabolites were significantly reduced (P≤0.05)as compared to Treatment 1. Combined treatments of low-fat ice cream supplemented with B. lactis Bb-12 and KMN (Treatment 4) contributed to a further significant diminution (P≤0.05) in toxic compounds from previous treatments (~0.3 g/L and ~0.7-0.9 g/L reductions in ammonia and total BA compared to Treatment 1, respectively), although levels were still higher than basal condition (control).This may be due to the inhibitory effect of probiotic and/or KMN on the growth of BA producing bacteria.Accumulation was also greater in the distal colon vessel of the gut reactor, regardless of treatment conditions, resulting from prolonged fermentation similarly observed in the study of Chaikham and Rattanasena (2017) while total BA levels remained mostly unchanged compared to baseline conditions.Additionally, Mäkeläinen, Ottman, Forssten et al. (2010) showed that probiotic B. lactis Bi-07 supplementation alone, or in combination with polydextrose, significantly increased the production of cadaverine and spermine, while treatments of galacto-oligosaccharide showed complete suppression and reduction in the levels of specific BA in the colon simulations.In the report of Mäkivuokko et al. (2010), L. acidophilus NCFM TM plus lactitol lowered cadaverine, putrescine and total BA concentrations in the colon simulator.
While many species of probiotic bacteria are known for their therapeutic and dietetic effects, some strains of lactic acid bacteria and bifidobacteria also have the ability to decarboxylate amino acids into toxic nitrogenous metabolites (Walstra et al., 2006;Chalarampopoulos and Rastall, 2009;Buňková et al., 2011).Although the level of BA formation by bifidobacteria is relatively low, it is contrastive to preexisting beneficial dietary effects (Buňková et al., 2011).BA threatens the wellbeing of consumers by contributing to increased toxicity potential of dairy products (Lorencová et al., 2014).Chronic putrefaction from long -term protein fermentation in the colon increases the risks of colon cancer through secretions of ammonia and biogenic amines, which encourage neoplastic growth of colon epithelium and stimulate the production of cocarcinogenic phenols (Ouwehand et al., 2005).Hence, measures to reduce secretion of toxic nitrogenous compounds and bacterial proteolytic activity must be undertaken to confirm health benefits upon administration of probiotic and KMN supplemented lowfat ice cream.

Conclusion
Our findings demonstrated significant modulations of the simulated colon microbiota following feeding of the human gut reactor with KMN and B. lactis Bb-12 supplemented low-fat ice cream.The formation of beneficial SCFA (acetic, butyric and propionic acids) increased, along with lactic acid concentrations in both proximal and distal colon vessels, by Treatments 2-4 as compared to Treatment 1.Both  suppressed the ammonia formation and BA formation in the colon.The greater increase in populations of bifidobacteria and lactobacilli, along with declination in the levels of clostridia and fecal coliforms, were also  Values are expressed as mean values ± standard deviation (n = 5).Means in the same rows with the same lowercase letters and means in the same columns with the same uppercase letters indicate no significant difference (P > 0.05).
observed in combined treatments of KMN and B. lactis Bb-12 compared to control, pure low-fat ice cream and separate supplementations of the latter.Changes in colon microbial diversity were supported by the formation of new patterns from generated PCR-DCGE fingerprints.In summary, KMN was able to boost the effects of probiotic-low-fat ice cream in improving the gut microbiota and their metabolites.
General colon microbial composition and population profiles were analyzed via PCR-DGCE fingerprinting of extracted bacterial DNA at Day 7 after feeding (Figure1).Feeding of the gut reactor with pure low-fat ice cream, probiotic-supplemented and KMN supplemented low-fat ice cream has led to clear differences in PCR-DCGE profile patterns compared to the basal condition.While differing band intensities and formation of new band positions were observed in all treatments on the 7 th day, the most drastic changes in band patterns occurred following treatment with combined supplementation of B. lactis Bb-12 and KMN in low-fat ice cream (Treatment 4).With reference to the treatment, intensities of bacterial rRNA also clearly decreased in zone A, while some bands in zone B were intensified.Generation of such profiles validated the synergism of combined effects of KMN and B. lactis Bb-12 supplementation on low-fat ice cream in modulating the gut microbiota.Patterns were comparable to PCR-DCGE profiles in the study of probiotic supplementations of low -fat ice cream involving a human gut reactor by Chaikham and Rattanasena (2017), where treatments of L. casei 01 and L. acidophilus LA5 strains resulted in considerable beneficial adjustments of the large intestinal microbial ecosystem.According to Bianchi et al. (2014), synbiotic vegetable beverages were found to induce changes in the composition and structure of microbial communities, especially lactobacilli, within stimulated colon compartments as observed from the increased number of bands in PCR-DCGE fingerprints.Similarly, Van Zanten et al. (2012) found that synbiotic combinations of B. lactis Bl-04 with melibiose, xylobiose, raffinose and maltotriose were able to shift predominant gut bacteria populations and increase their SCFA production in the colonic model system, thus may potentially be capable of manipulating the human gut microbiome.Synbiotic yogurt containing B. lactis Bb-12 and inulin could increase levels of bifidobacteria and reduce clostridia populations in a study of the human intestine by Palaria et al. (2012), using real-time PCR amplification (RT-PCR) techniques to quantify specific bacterial populations before and after yogurt treatment.
Figure 1.PCR-DGGE patterns of general colon bacteria obtained from proximal colon (PC) and distal colon (DC) compartments after feeding with different compositions of lowfat ice creams.

Table 2 .
Differences in feeding compositions of low-fat ice cream.

Table 3 .
Quantities of short-chain fatty acids and lactic acid in colon vessels after supplementation with difference treatments of low-fat ice cream.

Table 4 .
.DeValues are expressed as mean values ± standard deviation (n = 5).Means in the same rows with the same lowercase letters and means in the same columns with the same uppercase letters indicate no significant difference (P > 0.05).Microflora communities in colon vessels after supplementation with difference treatments of low-fat ice cream.

Table 5 .
Concentrations of ammonia and biogenic amines in colon vessels after supplementation with difference treatments of low-fat ice cream.