Preclinical Models of Anal Cancer Combined-Modality Therapy.

INTRODUCTION: There have been no significant changes in anal cancer treatment options in 4 decades. In this study, we highlight two preclinical models designed to assess anal cancer treatments. MATERIALS AND METHODS: Transgenic K14E6/E7 mice were treated with 7, 12-dimethylbenz(a)anthracene until anal tumors developed. Mice were treated with localized radiation in addition to chemotherapy (combined-modality therapy [CMT]) and compared to no treatment control (NTC). K14E6/E7 mouse anal spheroids with and without Pik3ca mutations were isolated and treated with vehicle, LY3023414 (LY3) (a drug previously shown to be effective in cancer prevention), CMT, or CMT + LY3. RESULTS: In the in vivo model, there was a significant increase in survival in the CMT group compared to the NTC group (P = 0.0392). In the ex vivo model, there was a significant decrease in the mean diameter of CMT and CMT + LY3-treated spheroids compared to vehicle (P ≤ 0.0001). For LY3 alone compared to vehicle, there was a statistically significant decrease in spheroid size in the K14E6/E7 group without mutation (P = 0.0004). CONCLUSIONS: We have provided proof of concept for two preclinical anal cancer treatment models that allow for the future testing of novel therapies for anal cancer.

Genetic inhibition of autophagy in a transgenic mouse model of anal cancer.

BACKGROUND: The dynamic role of autophagy in cancer development is a topic of considerable research and debate. Previously published studies have shown that anal cancer development can be promoted or prevented with the pharmacologic inhibition or induction, respectively, of autophagy in a human papillomavirus (HPV) mouse model. However, these results are confounded by the fact that the drugs utilized are known to affect other pathways besides autophagy. It has also been shown that autophagic inhibition occurs in the setting of HPV16 oncoprotein expression (E6 and E7) and correlates with increased susceptibility to anal carcinogenesis. MATERIALS AND METHODS: In this study, we employed a conditional, genetic, autophagic (Atg7) knockout mouse model to determine conclusively that autophagy has a role in anal cancer development, in the absence or presence of E6 and E7. RESULTS: In mice lacking both HPV16 oncogenes, knockout of autophagy followed by exposure to a carcinogen resulted in a tumor incidence of 40%, compared to 0% in mice treated with a carcinogen alone with an intact autophagic pathway (P = 0.007). In mice expressing either one or both HPV16 oncoproteins, the addition of genetic knockout of autophagy to carcinogen treatment did not lead to a significant difference in tumor incidence compared to carcinogen treatment alone, consistent with the ability of HPV oncogenes to inhibit autophagy in themselves. CONCLUSIONS: These results provide the first conclusive evidence for the distinct role of autophagy in anal carcinogenesis, and suggest that autophagy is a plausible target for therapies aimed at reducing anal dysplasia and anal cancer development.

The role of pharmacologic modulation of autophagy on anal cancer development in an HPV mouse model of carcinogenesis.

Autophagy is an intracellular, catabolic process that maintains cellular health. We examined the response of pharmacologic modulation of autophagy in an HPV mouse model of anal carcinogenesis. K14E6/E7 mice were treated with the topical carcinogen DMBA weekly and assessed for tumors over 20 weeks. Concurrently, they were given either chloroquine or BEZ235, to inhibit or induce autophagy, respectively. Time to tumor onset was examined. Immunofluorescence (IF) was performed for LC3ß and p62 to examine autophagy. All DMBA treated K14E6/E7 mice developed anal cancer, contrary to zero of the no DMBA treated mice. Chloroquine plus DMBA resulted in a significant decrease in the time to tumor onset compared to K14E6/E7 treated with DMBA. Only 40% BEZ235 plus DMBA treated mice developed anal cancer. Autophagic induction with DMBA and BEZ235, and autophagic inhibition with chloroquine were confirmed via IF. Anal carcinogenesis can be inhibited or induced via pharmacologic modulation of autophagy.

Dysregulation of Autophagy Contributes to Anal Carcinogenesis.

INTRODUCTION: Autophagy is an intracellular catabolic process that removes and recycles unnecessary/dysfunctional cellular components, contributing to cellular health and survival. Autophagy is a highly regulated cellular process that responds to several intracellular signals, many of which are deregulated by human papillomavirus (HPV) infection through the expression of HPV-encoded oncoproteins. This adaptive inhibitory response helps prevent viral clearance. A strong correlation remains between HPV infection and the development of squamous cell carcinoma (SCC) of the anus, particularly in HIV positive and other immunosuppressed patients. We hypothesize that autophagy is inhibited by HPV-encoded oncoproteins thereby promoting anal carcinogenesis (Fig 1). MATERIALS AND METHODS: HPV16 transgenic mice (K14E6/E7) and non-transgenic mice (FVB/N), both of which do not spontaneously develop anal tumors, were treated topically with the chemical carcinogen, 7,12-Dimethylbenz[a]anthracene (DMBA), to induce anal cancer. The anuses at different time points of treatment (5, 10, 15 and 20 weeks) were analyzed using immunofluorescence (IF) for two key autophagy marker proteins (LC3ß and p62) in addition to histological grading. The anuses from the K14E6/E7 mice were also analyzed for visual evidence of autophagic activity by electron microscopy (EM). To see if there was a correlation to humans, archival anal specimens were assessed histologically for grade of dysplasia and then analyzed for LC3ß and p62 protein content. To more directly examine the effect of autophagic inhibition on anal carcinogenesis, nontransgenic mice that do not develop anal cancer with DMBA treatment were treated with a known pharmacologic inhibitor of autophagy, chloroquine, and examined for tumor development and analyzed by IF for autophagic proteins. RESULTS: Histologically, we observed the progression of normal anoderm to invasive SCC with DMBA treatment in K14E6/E7 mice but not in nontransgenic, syngeneic FVB/N background control mice. With the development of low-grade dysplasia in the K14E6/E7 mice, there was an increase in both punctate LC3ß and p62 expression while EM revealed increased autophagosomes without evidence of autophagolysosomes. These observations are consistent with autophagy being inhibited at a later stage in the autophagic process. In contrast, in high-grade dysplasia and SCC in the DMBA-treated K14E6/E7 mice, there were decreased levels of p62 with a continued increase in punctate LC3ß expression by IF, while autophagolysosomes were seen on EM, consistent with the process of autophagy proceeded to completion. Similar findings, including histological grade dependent changes in LC3ß and p62 expression, were noted with human samples upon analysis of IF. Finally, with pharmacologic inhibition of autophagy in DMBA-treated, nontrangenic FVB/N mice, there was a significant increase in anal cancer development similar to that observed in DMBA- treated K14E6/E7 mice. CONCLUSION: Autophagic dysregulation is noted early on in HPV-associated anal carcinogenesis (low-grade dysplasia), with normalization of the autophagic process arising in late stages of HPV-associated anal carcinogenesis (high-grade dysplasia and invasive carcinoma).

Tissue-specific conditional PKCε knockout mice: a model to precisely reveal PKCε functional role in initiation, promotion and progression of cancer.

PKCε is a transforming oncogene and a predictive biomarker of various human cancers. However, a precise in vivo link of PKCε to cancer induction, progression and metastasis remain undefined. To achieve these goals, we generated tissue specific conditional PKCε knockout mice (PKCε-CKO) using cre-lox technology. Homozygous PKCε(LoxP/LoxP) mice have normal body weight and phenotype. To determine what effect loss of PKCε would have on the prostate, the PKCε(LoxP/LoxP) mice were bred to probasin cre (PB-Cre4+) mice which express cre specifically in the prostate epithelium of postnatal mice. Western blot and immunohistochemical analyses showed reduced levels of PKCε specifically in the prostate of PKCε-CKO mice. Histopathological analyses of prostate from both PKCε(LoxP/LoxP) and prostate PKCε-CKO mice showed normal pathology. To determine the functional impact of prostate specific deletion of PKCε on prostate tumor growth, we performed an orthotopic xenograft study. Transgenic adenocarcinoma of the mouse prostate (TRAMP) cells (TRAMPC1, 2×106) were implanted in the prostate of PKCε-CKO mice. Mice were sacrificed at 6th week post-implantation. Results demonstrated a significant (P<0.05) decrease in the growth of TRAMPC1 cells-derived xenograft tumors in PKCε-CKO mice compared to wild type. To determine a link of PKCε to ultraviolet radiation (UVR) exposure-induced epidermal Stat3 phosphorylation, PKCε(LoxP/LoxP) mice were bred to tamoxifen-inducible K14 Cre mice. PKCε deletion in the epidermis resulted in inhibition of UVR-induced Stat3 phosphorylation. In summary, our novel PKCε(LoxP/LoxP) mice will be useful for defining the link of PKCε to various cancers in specific organ, tissue, or cells.

Cholecystokinin expression in the ß-cell leads to increased ß-cell area in aged mice and protects from streptozotocin-induced diabetes and apoptosis.

Cholecystokinin (CCK) is a peptide hormone produced in the gut and brain with beneficial effects on digestion, satiety, and insulin secretion. CCK is also expressed in pancreatic ß-cells, but only in models of obesity and insulin resistance. Whole body deletion of CCK in obese mice leads to reduced ß-cell mass expansion and increased apoptosis. We hypothesized that islet-derived CCK is important in protection from ß-cell apoptosis. To determine the specific role of ß-cell-derived CCK in ß-cell mass dynamics, we generated a transgenic mouse that expresses CCK in the ß-cell in the lean state (MIP-CCK). Although this transgene contains the human growth hormone minigene, we saw no expression of human growth hormone protein in transgenic islets. We examined the ability of MIP-CCK mice to maintain ß-cell mass when subjected to apoptotic stress, with advanced age, and after streptozotocin treatment. Aged MIP-CCK mice have increased ß-cell area. MIP-CCK mice are resistant to streptozotocin-induced diabetes and exhibit reduced ß-cell apoptosis. Directed CCK overexpression in cultured ß-cells also protects from cytokine-induced apoptosis. We have identified an important new paracrine/autocrine effect of CCK in protection of ß-cells from apoptotic stress. Understanding the role of ß-cell CCK adds to the emerging knowledge of classic gut peptides in intraislet signaling. CCK receptor agonists are being investigated as therapeutics for obesity and diabetes. While these agonists clearly have beneficial effects on body weight and insulin sensitivity in peripheral tissues, they may also directly protect ß-cells from apoptosis.

Plumbagin Inhibits Prostate Carcinogenesis in Intact and Castrated PTEN Knockout Mice via Targeting PKCε, Stat3, and Epithelial-to-Mesenchymal Transition Markers.

Prostate cancer continues to remain the most common cancer and the second leading cause of cancer-related deaths in American males. The Pten deletions and/or mutations are frequently observed in both primary prostate cancers and metastatic prostate tissue samples. Pten deletion in prostate epithelium in mice results in prostatic intraepithelial neoplasia (PIN), followed by progression to invasive adenocarcinoma. The Pten conditional knockout mice [(Pten-loxp/loxp:PB-Cre4(+)) (Pten-KO)] provide a unique preclinical model to evaluate agents for efficacy for both the prevention and treatment of prostate cancer. We present here for the first time that dietary plumbagin, a medicinal plant-derived naphthoquinone (200 or 500 ppm) inhibits tumor development in intact as well as castrated Pten-KO mice. Plumbagin has shown no signs of toxicity at either of these doses. Plumbagin treatment resulted in a decrease expression of PKCε, AKT, Stat3, and COX2 compared with the control mice. Plumbagin treatment also inhibited the expression of vimentin and slug, the markers of epithelial-to-mesenchymal transition (EMT) in prostate tumors. In summary, the results indicate that dietary plumbagin inhibits growth of both primary and castration-resistant prostate cancer (CRPC) in Pten-KO mice, possibly via inhibition of PKCε, Stat3, AKT, and EMT markers (vimentin and slug), which are linked to the induction and progression of prostate cancer.

Topically applied Hsp90 inhibitor 17AAG inhibits UVR-induced cutaneous squamous cell carcinomas.

We present here that heat-shock protein 90 (Hsp90) inhibitor 17-(allylamino)-17-demethoxygeldanamycin (17AAG), when topically applied to mouse skin, inhibits UVR-induced development of cutaneous squamous cell carcinoma (SCC). In these experiments, DMSO:acetone (1:40 v/v) solution of 17AAG (500 nmol) was applied topically to mouse skin in conjunction with each UVR exposure (1.8 kJ m(-2)). The UVR source was Kodacel-filtered FS-40 sun lamps (approximately 60% UVB and 40% UVA). In independent experiments with three separate mouse lines (SKH-1 hairless mice, wild-type FVB, and protein kinase C epsilon (PKCɛ)-overexpressing transgenic FVB mice), 17AAG treatment increased the latency and decreased both the incidence and multiplicity of UVR-induced SCC. Topical 17AAG alone or in conjunction with UVR treatments elicited neither skin nor systemic toxicity. 17AAG-caused inhibition of SCC induction was accompanied by a decrease in UVR-induced (1) hyperplasia, (2) Hsp90ß-PKCɛ interaction, and (3) expression levels of Hsp90ß, Stat3, pStat3Ser727, pStat3Tyr705, pAktSer473, and matrix metalloproteinase (MMP). The results presented here indicate that topical Hsp90 inhibitor 17AAG is effective in prevention of UVR-induced epidermal hyperplasia and SCC. One may conclude from the preclinical data presented here that topical 17AAG may be useful for prevention of UVR-induced inflammation and cutaneous SCC either developed in UVR-exposed or organ transplant population.

α-Mangostin: a dietary antioxidant derived from the pericarp of Garcinia mangostana L. inhibits pancreatic tumor growth in xenograft mouse model.

AIMS: Pancreatic cancer (PC) is the most aggressive malignant disease, ranking as the fourth most leading cause of cancer-related death among men and women in the United States. In this study, we provide evidence of chemotherapeutic effects of α-mangostin, a dietary antioxidant isolated from the pericarp of Garcinia mangostana L. against human PC. RESULTS: The chemotherapeutic effect of α-mangostin was determined using four human PC cells (PL-45, PANC1, BxPC3, and ASPC1). α-Mangostin resulted in a significant inhibition of PC cells viability without having any effects on normal human pancreatic duct epithelial cells. α-Mangostin showed a dose-dependent increase of apoptosis in PC cells. Also, α-mangostin inhibited the expression levels of pNF-κB/p65Ser552, pStat3Ser727, and pStat3Tyr705. α-Mangostin inhibited DNA binding activity of nuclear factor kappa B (NF-κB) and signal transducer and activator 3 (Stat3). α-Mangostin inhibited the expression levels of matrix metallopeptidase 9 (MMP9), cyclin D1, and gp130; however, increased expression of tissue inhibitor of metalloproteinase 1 (TIMP1) was observed in PC cells. In addition, i.p. administration of α-mangostin (6 mg/kg body weight, 5 days a week) resulted in a significant inhibition of both primary (PL-45) and secondary (ASPC1) human PC cell-derived orthotopic and ectopic xenograft tumors in athymic nude mice. No sign of toxicity was observed in any of the mice administered with α-mangostin. α-Mangostin treatment inhibited the biomarkers of cell proliferation (Ki-67 and proliferating cell nuclear antigen [PCNA]) in the xenograft tumor tissues. INNOVATION: We present, for the first time, that dietary antioxidant α-mangostin inhibits the growth of PC cells in vitro and in vivo. CONCLUSION: These results suggest the potential therapeutic efficacy of α-mangostin against human PC.

Plumbagin, a medicinal plant (Plumbago zeylanica)-derived 1,4-naphthoquinone, inhibits growth and metastasis of human prostate cancer PC-3M-luciferase cells in an orthotopic xenograft mouse model.

We present here first time that Plumbagin (PL), a medicinal plant-derived 1,4-naphthoquinone, inhibits the growth and metastasis of human prostate cancer (PCa) cells in an orthotopic xenograft mouse model. In this study, human PCa PC-3M-luciferase cells (2 × 10(6)) were injected into the prostate of athymic nude mice. Three days post cell implantation, mice were treated with PL (2 mg/kg body wt. i.p. five days in a week) for 8 weeks. Growth and metastasis of PC-3M-luciferase cells was examined weekly by bioluminescence imaging of live mice. PL-treatment significantly (p = 0.0008) inhibited the growth of orthotopic xenograft tumors. Results demonstrated a significant inhibition of metastasis into liver (p = 0.037), but inhibition of metastasis into the lungs (p = 0.60) and lymph nodes (p = 0.27) was not observed to be significant. These results were further confirmed by histopathology of these organs. Results of histopathology demonstrated a significant inhibition of metastasis into lymph nodes (p = 0.034) and lungs (p = 0.028), and a trend to significance in liver (p = 0.075). None of the mice in the PL-treatment group showed PCa metastasis into the liver, but these mice had small metastasis foci into the lymph nodes and lungs. However, control mice had large metastatic foci into the lymph nodes, lungs, and liver. PL-caused inhibition of the growth and metastasis of PC-3M cells accompanies inhibition of the expression of: 1) PKCε, pStat3Tyr705, and pStat3Ser727, 2) Stat3 downstream target genes (survivin and Bcl(xL)), 3) proliferative markers Ki-67 and PCNA, 4) metastatic marker MMP9, MMP2, and uPA, and 5) angiogenesis markers CD31 and VEGF. Taken together, these results suggest that PL inhibits tumor growth and metastasis of human PCa PC3-M-luciferase cells, which could be used as a therapeutic agent for the prevention and treatment of human PCa.

Small-scale, semi-automated purification of eukaryotic proteins for structure determination.

A simple approach that allows cost-effective automated purification of recombinant proteins in levels sufficient for functional characterization or structural studies is described. Studies with four human stem cell proteins, an engineered version of green fluorescent protein, and other proteins are included. The method combines an expression vector (pVP62K) that provides in vivo cleavage of an initial fusion protein, a factorial designed auto-induction medium that improves the performance of small-scale production, and rapid, automated metal affinity purification of His8-tagged proteins. For initial small-scale production screening, single colony transformants were grown overnight in 0.4 ml of auto-induction medium, produced proteins were purified using the Promega Maxwell 16, and purification results were analyzed by Caliper LC90 capillary electrophoresis. The yield of purified [U-15N]-His8-Tcl-1 was 7.5 microg/ml of culture medium, of purified [U-15N]-His8-GFP was 68 microg/ml, and of purified selenomethione-labeled AIA-GFP (His8 removed by treatment with TEV protease) was 172 microg/ml. The yield information obtained from a successful automated purification from 0.4 ml was used to inform the decision to scale-up for a second meso-scale (10-50 ml) cell growth and automated purification. 1H-15N NMR HSQC spectra of His8-Tcl-1 and of His8-GFP prepared from 50 ml cultures showed excellent chemical shift dispersion, consistent with well folded states in solution suitable for structure determination. Moreover, AIA-GFP obtained by proteolytic removal of the His8 tag was subjected to crystallization screening, and yielded crystals under several conditions. Single crystals were subsequently produced and optimized by the hanging drop method. The structure was solved by molecular replacement at a resolution of 1.7 A. This approach provides an efficient way to carry out several key target screening steps that are essential for successful operation of proteomics pipelines with eukaryotic proteins: examination of total expression, determination of proteolysis of fusion tags, quantification of the yield of purified protein, and suitability for structure determination.

Plant Trials of Bacon Made with Lactic Acid Bacteria, Sucrose and Lowered Sodium Nitrite.

Bacon prepared with 40 and 80 mg/kg (ppm) sodium nitrite, 0.7% sucrose and a culture of Pediococcus acidilactici (Wisconsin Process), and control bacon prepared with 120 ppm sodium nitrite and no added sucrose or bacterial culture were produced at three commercial bacon production plants. Sodium chloride, phosphate and sodium ascorbate (or sodium erythorbate) levels, as well as other processing conditions such as pumping rate, smokehouse temperature and time, forming and slicing conditions, were those normally used by each plant. Randomly selected samples of each lot were used for a challenge experiment with Clostridium botulinum (types A and B), with ca. 1,000 heat-shocked spores/g of bacon inoculated on each slice, vacuum packaged and incubated at 27°C. Samples were taken periodically up to 56 d of incubation and examined for the presence of botulinal toxin. The challenge experiment revealed that test bacon was substantially greater in antibotulinal properties than the control bacon. Residual nitrite levels of test bacon were lower than those of the control bacon, as were nitrosamines formed upon frying. Average N-nitrospyrrolidine level was 8.6 µg/kg (ppb) in the control, <2.7 ppb in the 80-ppm nitrite product, and <1.6 ppb in the 40-ppm nitrite product. This study indicates that bacon commercially prepared by the Wisconsin Process with 40 or 80 ppm sodium nitrite has a lesser risk of nitrosamine and botulinal toxin formation than bacon prepared with 120 ppm sodium nitrite and no added sucrose and lactic acid bacteria.

Sensory Characteristics of Reduced Nitrite Bacon Manufactured by the Wisconsin Process.

Bacon with a culture of lactic acid-forming bacteria, Pediococcus acidilactici , plus 0.7% sucrose and 40 or 80 mg sodium nitrite/kg (Wisconsin Process), and control bacon with 120 mg sodium nitrite/kg but no lactic acid bacteria and sucrose, were produced at three commercial bacon plants under production conditions. The bacon was stored under refrigeration for 5 to 8 wk, then subjected to sensory analyses by an experienced sensory panel. Quantitative descriptive visual analysis was performed on uncooked as well as cooked samples, and the cooked samples were served for quantitative descriptive sensory analysis. Results indicated that the test bacon with reduced amounts of sodium nitrite was as acceptable as the control bacon with no sugar and lactics, with the 80 mg/kg nitrite-bacon being the most preferred of all. These results and the results of botulinal challenge and nitrosamine tests indicate that the test process can be a satisfactory alternative to processing bacon by the conventional procedure with 120 mg sodium nitrite/kg.

Survival of Listeria monocytogenes During the Manufacture and Storage of Nonfat Dry Milk.

The ability of Listeria monocytogenes to survive in skim milk during spray drying and to persist in nonfat dry milk during storage was examined. Concentrated (30% solids) and unconcentrated skim milks were inoculated with ca. 105 to 106 L. monocytogenes /ml and spray dried (inlet temperature, 165 ± 2°C; outlet temperature 67 ± 2°C) to a moisture content of 3.6 to 6.4%. The nonfat dry milk was packaged in moisture-resistant film and stored at 25°C for up to 16 wk. A reduction of ca. 1 to 1.5 log10 L. monocytogenes /g occurred during the spray drying process, irrespective of whether the milk was concentrated or not before spray drying. The organism progressively died during storage at 25°C, with a >4-log10 CFU/g decrease occurring within 16 wk of storage.

Evaluation of the Bacteriological Safety of Low-Salt Miso.

Studies were done to evaluate the safety of three different low-salt (2.36 to 5.79% NaCl) misos inoculated with different bacterial pathogens. Clostridium botulinum types A and B (inoculum level of ca. 120 spores/g) did not produce toxin in any of the misos within 18 wk at 25°C. Staphylococcus aureus , Salmonella typhimurium and Yersinia enterocolitica (inoculum level of ca. 103 to 104 CFU/g) progessively died in all of the misos held at either 10 or 25°C. The miso samples, which were obtained from Japan (3.75 and 5.79% NaCl) and California (2.36% NaCl), had water activities of 0.843, 0.835 and 0.875, respectively, and pH values of 5.26, 5.30 and 4.73, respectively. Results indicate that low-salt misos with these properties are not likely to be bacteriological health risks.

Evaluation of the Microbiological Safety of Tempeh Made from Unacidified Soybeans.

Studies were done to evaluate the safety of tempeh made from unacidifed soybeans and inoculated with different bacterial pathogens. Pathogens were added to either the soybeans before fermentation by Rhizopus oligosporus or the tempeh after fermentation and steaming. In the latter method, the inoculated products were incubated at several different temperatures (5, 10, 15 and 25°C). Clostridium botulinum (types A and/or B) toxin was produced in 2 d during the fermentation and within 5 d at 25°C or 4 wk at 15°C in tempeh inoculated and incubated in vacuum packages after fermentation and steaming. Staphylococcus aureus grew very well (>6-log10 CFU/g increase) in 2 d during the fermentation, and grew from ca. 103 CFU/g to 108 CFU/g in 7 d at 25°C and 21 d at 15°C in tempeh inoculated after fermentation and steaming. Staphylococcal enterotoxins were detected in some of these samples. Salmonella typhimurium also grew well during the fermentation (>6-log10 CFU/g increase in 1 d), but grew relatively slowly at 25 and 15°C in tempeh inoculated after fermentation and steaming. Yersinia enterocolitica grew very well (>6-log10 CFU/g increase) in 1 d during the fermentation, and also grew well in tempeh inoculated after fermentation and steaming, with a >6 log10 CFU/g increase in 2 d at 25 or 15°C and 5 d at 10°C. Results of these studies indicate the need for maintaining: (a) a high level of sanitary practices during production and (b) good refrigeration (≤5°C) of the product following fermentation until it is used.